Pyrrolidine ester derivatives with oxytocin modulating activity

ABSTRACT

The present invention is related to the use of pyrrolidine esters of formula (I) for the treatment and/or prevention of premature labor, premature birth and dysmenorrhea. In particular, the present invention is related to the use of pyrrolidine esters of formula (I) to modulate, notably to antagonise the oxytocin receptor. The present invention is furthermore related to novel pyrrolidine esters. X is selected from the group consisting of CR 6 R 7 , NOR 6 , NNR 6 R 7 ; R is selected from the group comprising or consisting of C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, saturated or unsaturated 3-8-membered cycloalkyl which may contain 1 to 3 heteroatoms selected of N, O, S, aryl, heteroaryl, C 1 -C 6 -alkyl aryl, C 1 -C 6 -alkyl heteroaryl. R 1  is selected from the group comprising or consisting of C 1 -C 6 -alkyl, C 2 -C 6 -alkenyl, C 2 -C 6 -alkynyl, aryl, heteroaryl, 3-8-membered cycloalkyl, acyl, C 1 -C 6 -alkyl aryl, C 1 -C 6 -alkyl heteroaryl, said cycloalkyl or aryl or heteroaryl groups may be fused with 1-2 further cycloalkyl or aryl or heteroaryl group.

FIELD OF THE INVENTION

[0001] The present invention is related to the use of pyrrolidine estersof formula (I) for the treatment and/or prevention of premature labor,premature birth and dysmenorrhea. In particular, the present inventionis related to the use of pyrrolidine esters of formula (I) to modulate,notably to antagonise, the oxytocin receptor. The present invention isfurthermore also related to novel pyrrolidine esters.

BACKGROUND OF THE INVENTION

[0002] Oxytocin (OT) is a peptide hormone and causes the contraction ofthe uterus of mammals during labor. The corresponding oxytocin receptorbelongs to the family of G-protein-coupled receptors and is similar toV_(1a) and V₂ vasopressin receptors. OT receptors increase dramaticallyduring the course of pregnancy. The concentration of OT receptors hasbeen shown to correlate with spontaneous uterine activity (M. Maggi etal. J. Clin. Endocrinol Metabol; 70; 1142, 1990). Premature labor,though, and premature birth is undesired as it represents a major causeof perinatal morbidity and mortality. Hence, the management of pretermlabor represents a significant problem in the field of obstetrics.

[0003] In recent years, strong evidence has accumulated indicating thatthe hormone oxytocin plays a major role in initiating labor in mammals,notably in humans. Thereby, it is assumed that oxytocin exerts saideffect in a direct as well as an indirect way, by contracting theuterine myometrium and by enhancing the synthesis and release ofcontractile prostaglandins from the uterine endometrium/decidua. Theseprostaglandins may further-more play a role in the cervical ripeningprocess. This “up-regulation” of oxytocin receptors and increaseduterine sensitivity seems to be due to trophic effects of rising plasmalevels of estrogen towards term. By down-regulating oxytocin, it isexpected that both the direct (contractile) and indirect (increasedprostaglandin synthesis) effects of oxytocin on the uterine could beblocked. An oxytocin modulator, e.g. blocker or antagonists would likelybe more efficacious for treating preterm labor than current regimens.Moreover, as oxytocin at term has only an effect on the uterus, such anoxytocin modulator would have only few or no side efect.

[0004] A further condition being related to oxytocin is dysmenorrhea,which is characterised by cyclic pain associated with menses duringovulatory cycles. Said pain is believed to result from uterinecontractions and ischemia, probably mediated by the effect ofprostaglandins produced in the secretory endometrium. By blocking boththe indirect and direct effects of oxytocin on the uterus, an oxytocinantagonist is believed more efficacious for treating dysmenorrhea thancurrent regimens.

[0005] Some agents counteracting the action of oxytocin (OT) arecurrently used in clinical studies. Such tocolytic agents (i.e.uterine-relaxing agents) include beta-2-adrenergic agonists, magnesiumsulfate and ethanol. The leading beta-2-adrenergic agonists isRitodrine, which causes a number of cardiovascular and metabolic sideeffects, including tachycardia, increased renin secretion, hyperglycemiaand reactive hypoglycemia in the infant. Further beta-32-adrenergicagonists, including terbutaline and albuterol have side effcts similarto those of ritodrine. Magnesium sulfate at plasma concentrations abovethe therapeutic range of 4 to 8 mg/dL can cause inhibition of cardiacconduction and neuromuscular transmission, respiratory depression andcardiac arrest, thus making this agent unsuitable when renal function isimpaired. Ethanol is as effective as ritodrine in preventing prematurelabor, but it does not produce a corresponding reduction in theincidence of fetal respiratory distress that administration of ritodrinedoes.

[0006] The principal drawback to the use of peptide antagonistsincluding also atosiban is the problem of low oral bioavailabilityresulting from intestinal degradation. Hence, they are administeredparenterally.

[0007] The development of non-peptide ligands for pepetide hormonereceptors are expected to overcome this problem. The first to reportsmall molecule selective oxytocin antagonists was Merck. Apart fromcyclic hexapeptides, Merck suggested indanylpiperidines andtolylpiperazines as orally deliverable OT antagonists (Evans et al. J.Med. Chem., 35, 3919 (1992). In WO 96/22775 and U.S. Pat. No. 5,756,497Merck reported benzoxazinylpiperidines or benzoxazinones as OT receptorantagonists.

[0008] The objective of this invention is to provide substances whichmore effectively down-regulate—up to antagonizing—the function of OT indisease states in animals, preferably mammals, especially in humans. Itis another purpose of this invention to provide a method of antagonizingthe functions of oxytocin in disease states of mammals. It is also anobjective of the present invention to provide small molecule chemicalcompounds for the modulation, preferably the down-regulation or evenantagonisation of the oxytocin receptor. Moreover, it is an objective ofthe present invention to provide methods for preparing said smallmolecule chemical compounds. It is furthermore an objective of thepresent invention to provide a new category of pharmaceuticalformulations for the treatment of preterm labor and dysmenorrhea, and/ordiseases mediated by the oxytocin receptor. It is finally an objectiveof the present invention to provide a method of treating and/orpreventing disorders mediated by the oxytocin receptor, like pretermlabor and dysmenorrhea by antagonising the binding of oxytocin to itsreceptor.

SUMMARY OF THE INVENTION

[0009] The present invention relates to use of pyrrolidine esters offormula (I) for the treatment and/or prevention of premature labor,premature birth and dysmenorrhea. In particular, the present inventionis related to the use of pyrrolidine esters of formula (I) to modulate,notably to antagonise the oxytocin receptor. The present invention isfurthermore related to novel pyrrolidine esters.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The aforementioned objectives have been met according to theindependent claims. Preferred embodiments are set out within thedependent claims which are incorporated herein.

[0011] The following paragraphs provide definitions of the variouschemical moieties that make up the compounds according to the inventionand are intended to apply uniformly throughout the specification andclaims unless an otherwise expressly set out definition provides abroader definition.

[0012] “C₁-C₆-alkyl” refers to monovalent alkyl groups having 1 to 6carbon atoms. This term is exemplified by groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl and thelike.

[0013] “Aryl” refers to an unsaturated aromatic carbocyclic group offrom 6 to 14 carbon atoms having a single ring (e.g. phenyl) or multiplecondensed rings (e.g. naphthyl). Preferred aryl include phenyl,naphthyl, phenantrenyl and the like.

[0014] “C₁-C₆-alkyl aryl” refers to C₁-C₆-alkyl groups having an arylsubstituent, including benzyl, phenethyl and the like.

[0015] “Heteroaryl” refers to a monocyclic heteroaromatic, or a bicyclicor a tricyclic fused-ring heteroaromatic group. Particular examples ofheteroaromatic groups include optionally substituted pyridyl, pyrrolyl,furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl,[2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl,isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl,imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl,quinazolinyl, pthalazinyl, quinoxalinyl, cinnolinyl, napthyridinyl,pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl,quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl,5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl,xanthenyl or benzoquinolyl.

[0016] “C₁-C₆-alkyl heteroaryl” refers to C₁-C₆-alkyl groups having aheteroaryl substituent, including 2-furylmethyl, 2-thienylmethyl,2-(1H-indol-3-yl)ethyl and the like.

[0017] “Alkenyl” refers to alkenyl groups preferably having from 2 to 6carbon atoms and having at least 1 or 2 sites of alkenyl unsaturation.Preferable alkenyl groups include ethenyl (—CH═CH₂), n-2-propenyl(allyl, —CH₂CH═CH₂) and the like.

[0018] “Alkynyl” refers to alkynyl groups preferably having from 2 to 6carbon atoms and having at least 1-2 sites of alkynyl unsaturation,preferred alkynyl groups include ethynyl (—C≡CH), propargyl (—CH₂C≡CH),and the like.

[0019] “Acyl” refers to the group —C(O)R where R includes H,“C₁-C₆-alkyl”, “aryl”, “heteroaryl”, “C₁-C₆-alkyl aryl” or “C₁-C₆-alkylheteroaryl”.

[0020] “Acyloxy” refers to the group —OC(O)R where R includes H,“C₁-C₆-alkyl”, “aryl”, “hetero-aryl”, “C₁-C₆-alkyl aryl” or “C₁-C₆-alkylheteroaryl”.

[0021] “Alkoxy” refers to the group —O—R where R includes “C₁-C₆-alkyl”or “aryl” or “heteroaryl” or “C₁-C₆-alkyl aryl” or “C₁-C₆-alkylheteroaryl”. Preferred alkoxy groups include by way of example, methoxy,ethoxy, phenoxy and the like.

[0022] “Alkoxycarbonyl” refers to the group —C(O)OR where R includes“C₁-C₆-alkyl” or “aryl” or “heteroaryl” or “C₁-C₆-alkyl aryl” or“C₁-C₆-alkyl heteroaryl”.

[0023] “Aminocarbonyl” refers to the group —C(O)NRR¹ where each R, R¹includes independently hydrogen or C₁-C₆-alkyl or aryl or heteroaryl or“C₁-C₆-alkyl aryl” or “C₁-C₆-alkyl heteroaryl”.

[0024] “Acylamino” refers to the group —NR(CO)R¹ where each R, R¹ isindependently hydrogen or “C₁-C₆-alkyl” or “aryl” or “heteroaryl” or“C₁-C₆-alkyl aryl” or “C₁-C₆-alkyl heteroaryl”.

[0025] “Halogen” refers to fluoro, chloro, bromo and iodo atoms.

[0026] “Sulfonyl” refers to group “—SO₂—R” wherein R is selected from H,“aryl”, “heteroaryl”, “C₁-C₆-alkyl”, “C₁-C₆-alkyl” substituted withhalogens e.g. an —SO₂—CF₃ group, “C₁-C₆-alkyl aryl” or “C₁-C₆-alkylheteroaryl”.

[0027] “Sulfoxy” refers to a group “—S(O)—R” wherein R is selected fromH, “C₁-C₆-alkyl”, “C₁-C₆-alkyl” substituted with halogens e.g. an—SO—CF₃ group, “aryl”, “heteroaryl”′, “C₁-C₆alkyl aryl” or “C₁-C₆-alkylheteroaryl”.

[0028] “Thioalkoxy” refers to groups —S—R where R includes “C₁-C₆-alkyl”or “aryl” or “heteroaryl” or “C₁-C₆-alkyl aryl” or “C₁-C₆-alkylheteroaryl”. Preferred thioalkoxy groups include thiomethoxy,thioethoxy, and the like.

[0029] “Substituted or unsubstituted”: Unless otherwise constrained bythe definition of the individual substituent given in the presentspecification, the above set out groups, like “alkyl”, “alkenyl”,“alkynyl”, “aryl” and “heteroaryl” etc. groups may optionally besubstituted with from 1 to 5 substituents selected from the groupconsisting of “C₁-C₆-alkyl”, “C₁-C₆-alkyl aryl”, “C₁-C₆-alkylheteroaryl”, “C₂-C₆-alkenyl”, “C₂-C₆-alkynyl”, primary, secondary ortertiary amino groups or quarternary ammonium moieties, “acyl”,“acyloxy”, “acylamino”, “aminocarbonyl”, “alkoxycar-bonyl”, “aryl”,“heteroaryl”, carboxyl, cyano, halogen, hydroxy, mercapto, nitro,sulfoxy, sulfonyl, alkoxy, thioalkoxy, trihalomethyl and the like.Alternatively said substitution could also comprise situations whereneighboring substituents have undergone ring closure, notably whenviccinal functional substituents are involved, thus forming e.g.lactams, lactons, cyclic anhydrides, but also acetals, thioacetals,aminals formed by ring closure for instance in an effort to obtain aprotective group.

[0030] “Pharmaceutically acceptable salts or complexes” refers to saltsor complexes of the below-identified compounds of formula (I) thatretain the desired biological activity. Examples of such salts include,but are not restricted to acid addition salts formed with inorganicacids (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid, and the like), and salts formed withorganic acids such as acetic acid, oxalic acid, tartaric acid, succinicacid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoicacid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalene sulfonic acid, naphthalene disulfonic acid, andpoly-galacturonic acid. Said compounds can also be administered aspharmaceutically acceptable quaternary salts known by a person skilledin the art, which specifically include the quarternary ammonium salt ofthe formula —NR,R′,R″⁺Z⁻, wherein R, R′, R″ is independently hydrogen,alkyl, or benzyl, and Z is a counterion, including chloride, bromide,iodide, —O-alkyl, toluenesulfonate, methylsulfonate, sulfonate,phosphate, or carboxylate (such as benzoate, succinate, acetate,glycolate, maleate, malate, fumarate, citrate, tartrate, ascorbate,cinnamoate, mandeloate, and diphenylacetate).

[0031] “Pharmaceutically active derivative” refers to any compound thatupon administration to the recipient, is capable of providing directlyor indirectly, the activity disclosed herein.

[0032] “Enantiomeric excess” (ee) refers to the products that areobtained by an asymmetric synthesis, i.e. a synthesis involvingnon-racemic starting materials and/or reagents or a synthesis comprisingat least one enantioselective step, whereby a surplus of one enantiomerin the order of at least about 52% ee is yielded. In the absence of anasymmetric synthesis, racemic products are usually obtained that dohowever also have the inventive set out activity-as OT-R antagonists.

[0033] It was now found that pyrrolidine ester derivatives according toformula (I) are useful for the treatment and/or prevention of pretermlabor, premature birth and dysmenorrhea of mammals and in particular ofhumans. Specifically, the pyrrolidine ester derivatives according toformula (I) are useful for the treatment and/or prevention ofdisorders-related to the oxytocin function, i.e. disorders that aremediated by the oxytocin receptor. Preferably, the compounds of formula(I) are suitable to modulate, in particular to down-regulate the OT-Rfunction and more specifically to antagonise the oxytocin receptor. Whenthe oxytocin receptor is bound by the compounds according to formula(I), oxytocin is antagonised by being blocked from its receptor and istherefore unable to exert its biologic or pharmacological effects.

[0034] The compounds being suitable for the treatment and/or preventionof preterm labor, premature birth and dysmenorrhea are those of formula(I).

[0035] Formula (I) also comprises geometrical isomers, optically activeforms like enantiomers, diastereomers and racemate forms, as well aspharmaceutically acceptable salts thereof.

[0036] Preferred pharmaceutically acceptable salts of the compound I,are acid addition salts formed with pharmaceutically acceptable acidslike hydrochloride, hydrobromide, sulfate or bisulfate, phosphate orhydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate,lactate, citrate, tartrate, gluconate, methanesulfonate,benzenesulfonate, and para-toluenesulfonate salts.

[0037] In said formula (I), X is selected from the group consisting ofCR⁶R⁷, NOR⁶, NNR⁶R⁷.

[0038] R is selected from the group comprising or consisting ofunsubstituted or substituted C₁-C₆ alkyl, unsubstituted or substitutedC₂-C₆ alkenyl, unsubstituted or substituted C₂-C₆ alkynyl, unsubstitutedor substituted saturated or unsaturated 3-8-membered-cycloalkyl whichmay contain 1 to 3 heteroatoms selected of N, O, S, unsubstituted orsubstituted aryl, unsubstituted or substituted heteroaryl, unsubstitutedor substituted C₁-C₆-alkyl aryl, unsubstituted or substitutedC₁-C₆-alkyl heteroaryl.

[0039] R¹ is selected from the group comprising or consisting ofunsubstituted or substituted C₁-C6-alkyl, unsubstituted or substitutedC₂-C₆-alkenyl, unsubstituted or substituted C₂-C₆alkynyl, unsubstitutedor substituted aryl, unsubstituted or substituted heteroaryl,unsubstituted or substituted saturated or unsaturated 3-8-memberedcycloalkyl, acyl, unsubstituted or substituted C₁-C₆-alkyl aryl,unsubstituted or substituted C₁-C₆-alkyl heteroaryl, said cycloalkyl oraryl or heteroaryl groups may be fused with 1-2 further cycloalkyl oraryl or heteroaryl group.

[0040] R², R³, R⁴ and R⁵ are independently selected from each other fromthe group consisting of hydrogen, halogen, C₁-C₆-alkyl. Preferably theyare all hydrogen.

[0041] R⁶ and R⁷ are independently selected from the group comprising orconsisting of hydrogen, unsubstituted or substituted C₁-C₆ alkyl,unsubstituted or substituted C₂-C₆ alkenyl, unsubstituted or substitutedC₂-C₆ alkynyl, unsubstituted or substituted alkoxy, unsubstituted orsubstituted thioalkoxy, halogen, cyano, nitro, acyl, alkoxycarbonyl,aminocarbonyl, unsubstituted or substituted saturated or unsaturated3-8-membered cycloalkyl which may contain 1 to 3 heteroatoms selected ofN, O, S, unsubstituted or substituted aryl, unsubstituted or substitutedheteroaryl, unsubstituted or substituted Ct-C₆-alkyl aryl, unsubstitutedor substituted C₁-C₆-alkyl heteroaryl. Alternatively, R⁶, R⁷ could formtogether with the N atom to which they are attached a 3-8 memberedsubstituted or unsubstituted, saturated or unsaturated heterocyclic ringwhich may contain 1-2 further heteroatoms selected from N, S and O andwhich is optionally fused with an aryl, heteroaryl or 3-8 memberedsaturated or unsaturated cycloalkyl ring.

[0042] Compounds of formula (I)—wherein R is H or alkyl—are disclosed inWO 99/52868. The compounds claimed therein are said to be inhibitors ofmetalloproteases.

[0043] Preferred pyrrolidine derivatives are those compounds accordingto formula (I) wherein R is an unsubstituted or substituted C₁-C₆ alkyl.

[0044] Particularly preferred pyrrolidine derivatives are thosecompounds according to formula (I) wherein X is NOR⁶, and R⁶ is selectedfrom the group consisting of H, unsubstituted or substituted C₁-C₆alkyl, unsubstituted or substituted C₂-C₆ alkenyl, unsubstituted orsubstituted C₂-C₆ alkynyl, unsubstituted or substituted acyl,unsubstituted or substituted aryl, unsubstituted or substitutedheteroaryl, unsubstituted or substituted saturated or unsaturated3-8-membered cycloalkyl, unsubstituted or substituted C₁-C₆-alkyl aryl,unsubstituted or substituted C₁-C₆-alkyl heteroaryl, said cycloalkyl oraryl or heteroaryl groups may be fused with 1-2 further cycloalkyl oraryl or heteroaryl groups. Particularly preferred R⁶ is H or CH₃.

[0045] More preferred groups R¹ are substituted or unsubstitutedC₁-C₆-alkyl, substituted or unsubstituted C₂-C₆-alkenyl, unsubstitutedor substituted C₂-C₆-alkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, saturated or unsaturated3-8-membered substituted or unsubstituted cycloalkyl and still morepreferred R¹ are substituted or unsubstituted C₁-C₆-alkyl or substitutedor unsubstituted aryl. A particularly preferred substituent R¹ is asubstituted or unsubstituted biphenyl.

[0046] According to a most preferred embodiment, the pyrrolidinederivatives according to formula I are those wherein X is NOR⁶, R⁶ is H,a C₁-C₆-alkyl, e.g. a methyl group, or aryl or C₁-C₆-alkyl aryl groupand R¹ is a C₁-C₆-alkyl or aryl or C₁-C₆-alkyl aryl group. Even morepreferred are those pyrrolidine derivatives, wherein X is NOR⁶, R⁶ ismethyl, R is an unsubstituted or substituted C₁-C₆-alkyl group, e.g. amethyl group and R¹ is a substituted or unsubstituted biphenyl.

[0047] The compounds of formula (I) may contain one or more asymmetriccenters and may therefore exist as enantiomers or diastereoisomers. Itis to be understood that the invention includes both mixtures andseparate individual isomers or enantiomers of the compounds of formula(I). In a particularly preferred embodiment the pyrrolidine derivativesaccording to formula (I) are obtained in an enantiomeric excess of atleast 52% ee, preferably of at least 92-98% ee. Also, E/Z isomers withregard to pyrrolidine derivatives having residues X being ═CR⁶R⁷ wherebyboth R⁶R⁷ are different from each other, and/or with regard topyrrolidine derivatives having residues X being ═NOR⁶ or ═NNR⁶R⁷ arecomprised by the present invention.

[0048] A further aspect of the present invention is related to the useof the pyrrolidine derivatives according to formula (I) for thepreparation of pharmaceutical compositions for the treatment and/orprevention of premature labor, premature birth, for stopping labor priorto cesarean delivery and dysmenorrhea. Preferably, the compoundsaccording to formula (I) are suitable for the modulation of the OTfunction, thus specifically allowing the treatment and/or prevention ofdisorders which are mediated by the oxytocin receptor. Said treatmentinvolves the modulation—notably the down regulation or theantagonisation—of the oxytocin receptor.

[0049] Still a further aspect of the present invention is related to theactually novel pyrrolidine compounds of formula (I). Said compounds havethe formula (I′)

[0050] In formula (I′), R is selected from C₁-C₆ alkyl, C₁-C₆ alkylaryl, C₁-C₆ alkyl heteroaryl, 3-8-membered cycloalkyl. R¹ is selectedfrom an unsubstituted or substituted 1,1′-biphenyl, pyridinyl-phenyl orpyrimidinyl-phenyl group.

[0051] More preferred is R being a C₁-C₄ alkyl, i.e. a methyl, ethyl,propyl orbutyl or group, most preferred a methyl group.

[0052] More preferred R¹ is a 1,1′-biphenyl group which is substitutedby 1 or 2 moieties selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ alkoxy, halogen, CN. Most preferred is a methyl group.

[0053] Specific compounds of formula (I′) are the following:

[0054] Methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate

[0055] Methyl(2S,4EZ)-1-([1,1′-biphenyl]-4-ylcarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0056] Methyl(2S,4E)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]4-yl)carbonyl]-2-pyrrolidinecarboxylate

[0057] Methyl(2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate

[0058] Methyl(2S,4EZ)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0059] Methyl(2S,4)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0060] Methyl(2S,4EZ)-1-[(2′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0061] Methyl(2S,4EZ)-1-[(2′-cyano[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0062] Methyl(2S,4EZ)-4-(methoxyimino)-1-{[2-(trifluoromethyl)[1,1′-biphenyl]-4-yl]carbonyl}-2-pyrrolidinecarboxylate

[0063] Methyl(2S,4EZ-1-[(2′-methoxy[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0064] Methyl(2S,4EZ)-1-[(2′,6′-dimethyl[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0065] Methyl(2S,4EZ)-1-[(2′,3-dimethyl[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0066] Methyl(2S,4EZ)-1-[(3-methyl[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0067] Methyl(2S,4EZ)-1-[(3′,4′-dichloro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0068] Ethyl(2S,4EZ)-1-([1,1′-biphenyl]-4-ylcarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0069] sec-butyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate

[0070] Cyclopentyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate

[0071] Methyl(2S,4EZ)-1-[(4′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0072] Methyl(2S,4EZ)-4-(methoxyimino)-1-[4-(5-pyrimidinyl)benzoyl]-2-pyrrolidinecarboxylate

[0073] Methyl(2S,4EZ)-4-(methoxyimino)-1-[4-(2-pyridinyl)benzoyl]-2-pyrrolidinecarboxylate

[0074] Methyl(2S,4EZ)-4-(methoxyimino)-1-[4-(3-methyl-2-pyridinyl)benzoyl]-2-pyrrolidinecarboxylate

[0075] The pyrrolidine derivatives exemplified in this invention can beprepared from readily available starting materials using the followinggeneral methods and procedures. It will be appreciated that wheretypical or preferred experimental conditions (i.e. reactiontemperatures, time, moles of reagents, solvents, etc.) are given, otherexperimental conditions can also be used unless otherwise stated.Optimum reaction conditions may vary with the particular reactants orsolvents used, but such conditions can be determined by one skilled inthe art by routine optimisation procedures.

[0076] Generally, the pyrrolidine derivatives according to the generalformula (I) could be obtained by several processes, using bothsolution-phase and solid-phase chemistry protocols. Depending on thenature of the R- and X-m oieties, certain synthtic approaches will, insome instances, be preferred over others, and it is assumed that thechoice of the most suitable process will be evident to the practitionerskilled in the art.

[0077] According to one process, pyrrolidine derivatives according tothe general formula (I), whereby the substituents X, R and R¹⁻⁵ are asabove defined, are prepared from the corresponding suitably N-protected4-substituted pyrrolidine derivatives II, whereby the substituents X, Rand R¹⁻⁵ are as above defined, by solution-phase chemistry protocolssuch as described in the Examples and shown in Scheme 1, below. Removalof the N-protecting group of II, using an appropriate deprotection agent(e.g. TFA, piperidine, H₂/Pd/C) under standard conditions forN-deprotection well known to the person skilled in the art, producesderivatives of formula (III). These can be treated with acylating agentsof general formula (IV), whereby the substituent R¹ is as above defined,while Y could be any appropriate leaving group. Preferred acylatingagents IV are acid chlorides (IVa), used in conjunction with a tertiaryamine base, or carboxylic acids (IVb), used in conjunction with anappropriate peptide coupling agents, such as e.g. DIC, EDC, TBTU, DECP,or others, to yield the pyrrolidine ester products of general formula(I), with X, R and R¹⁻⁵ being as above defined.

[0078] Pyrrolidine ester compounds of formula (II), whereby thesubstituents X, R and R¹⁻⁵ are as above defined, are obtained from thecorresponding pyrrolidine carboxylic acids V, and alcohols VI, accordingto any of the standard methods well know to the person skilled in theart for transforming a carboxylic acid into an ester, e.g. thosedescribed in the Examples and shown in Scheme 2. The choice of the bestreagent and reaction conditions will depend on the nature of the X- andR-groups, and of the N-protection group, as will be obvious to thepractitioner skilled in the art.

[0079] Intermediate compounds of formula V, whereby the substituent X isCR⁶R⁷, and R⁶ and R⁷ are as above defined (i.e. compounds of formulaVa), may be prepared from compounds of general formula VI by Wittig-typereactions with anions of phosphoranes such as Vila and/or ofphosphonates such as VIIb, followed by saponification of the esterfunction using standard synthetic techniques, as hereinafter describedin the Examples and shown in scheme 3.

[0080] Intermediate compounds of formula V, wherein the substituent X isNOR⁶ or NNR⁶R⁻⁷, and R⁶ and R⁷ are as above defined (i.e. compounds offormula Vb and Vc), may be prepared from compounds of general formula(IX) by reaction with substituted hydroxylamines Xb and/or substitutedhydrazines and/or hydrazides Xc using standard synthetic techniques ashereinafter described in the Examples and shown in Scheme 4. Compoundsof formula Xa are commercially available or prepared by standardsynthetic techniques as hereinafter described in the Examples.

[0081] The intermediate compounds of general formulae VI and/or IX maybe prepared from commercially available, suitably N-protected (e.g. Boc)4-hydroxyprolines XI, by a reaction sequence consisting of oxidationand, if appropriate, methylation, using standard synthetic techniques ashereinafter described in the Examples and illustrated in Scheme 5.

[0082] A further, alternative approach of preparing the compounds of thepresent invention is depicted in Scheme 6. Following to this process thepyrrolidine derivatives—whereby the substituents X, R and R¹⁻⁵ are asabove defined—are prepared from compounds of formula XII, using thesynthetic techniques as outlined in Schemes 3 and 4. As further shown inScheme 6, compounds of formula XII may be obtained from compounds offormula (Ia) through transformation of the methyloxime into the ketonemoiety, e.g. under mild hydrolysis conditions as described hereinafterin the Examples. This present synthetic strategy is most preferred whereX is NOH or NNR⁶R⁷, whereby the substituents R⁶ and R⁷ are as abovedefined.

[0083] According to yet another process, pyrrolidine ester derivativesof general formula (I)^(X) can be interconverted (transformed) topyrrolidine ester derivatives of general formula (I)^(Y) by a reactionsequence comprising saponification and re-esterification with alcoholsVI^(Y), using standard conditions well known to the person skilled inthe art, as described hereinafter in the Examples and illustrated inScheme 7.

[0084] R^(x) and R^(y) are as above defined for R, but are differentfrom each other for the purpose of the trans-esterification.

[0085] According to yet another process, pyrrolidine ester derivativesaccording to the general formula (I), whereby the substituents X, R andR¹⁻⁵ are as above defined, are prepared from the corresponding suitablyN-protected 4-substituted pyrrolidine carboxylic acid derivatives V,whereby the substituent X is above defined, by a solid-phase protocolsuch as described in the examples and shown in Scheme 8, below. TheN-Boc-protected 4-substituted pyrrolidine derivative V is reacted with aresin carrying a linker prone to cleavage by nucelophiles, e.g. withKaiser oxime resin, using standard carbodiimide-mediated couplingconditions well known to the practitioner skilled in the art.Boc-deprotection with dilute TFA in DCM, or with BF₃.OEt₂ in dilute HOAcin DCM, affords compounds of formula XVI. The latter compound can betreated with acylating agents of general formula (IV), whereby thesubstituent R¹ is as above defined, and Y could be an appropriateleaving group. Preferred acylating agents IV are acid chlorides (IVa),used in conjunction with a tertiary amine base, or carboxylic acids(IVb), used in conjunction with a peptide coupling agent, such as e.g.DIC, EDC, TBTU, DECP, or others, to yield products of general formulaXVII.

[0086] In order to obtain the final compounds of general formula (I),the linkage to the resin is cleaved by prolonged treatment with alcoholsVI, and a tertiary, non-nucleophilic amine base, such as TEA, DIEA, DBU,or others. The circles in Scheme 8 symbolize the resin beads to whichthe corresponding compounds are linked during the solid phase synthesis.Other derivatives of formula (I) are prepared using known modificationsto, or variations of, the Scheme 8 reaction sequence. Further to theabove mentioned Kaiser oxime resin, other suitable reagents, notablyresins, known to a person skilled in the art, could be employed for thesolid-phase synthesis of compounds of general formula (I).

[0087] The reaction sequences outlined in the above Schemes provideenantiomerically pure compounds of formula (I), if enantiomerically purestarting materials are used. (R)- as well as (S)-enantiomers can beobtained depending upon whether (R)- or (S)-forms of commerciallyavailable compounds of formulas IV, V, X, and/or VI were used as thestarting materials.

[0088] However, the reaction sequences outlined in the above Schemesusually provides mixtures of (E)- and (Z)-isomers with respect to thesubstituents on the exocyclic double bond of the pyrrolidine ring. Inall cases studied, these (E)/(Z)-isomers could be separated by standardchromatography techniques well known to the person skilled in the art,such as by reversed phase high-pressure liquid chromatography (HPLC) orsilica gel flash chromatography (FC). The assignment of the absoluteconfiguration of the exocyclic double bond was performed usingNMR-techniques well described in the literature as will be known to thepractitioner skilled in the art (for configurationnal assignments ofe.g. oxime functionalities, see e.g. E. Breitmaier, W. Voelter Carbon-13NMR Spectroscopy, 3rd Ed, VCH, 1987, p. 240).

[0089] According to a further general process, compounds of formula (I)can be converted to alternative compounds of formula (I), employingsuitable interconversion techniques such as hereinafter described in theExamples.

[0090] If the above set out general synthetic methods are not applicablefor obtaining compounds according to formula (I) and/or necessaryintermediates for the synthesis of compounds of formula (I), suitablemethods of preparation known by a person skilled on the art should beused. In general, the synthesis pathways for any individual compound offormula (I) will depend on the specific substitutents of each moleculeand upon the ready availability of intermediates necessary; again suchfactors being appreciated by those of ordinary skill in the art. For allthe protection, de-protection methods, see Philip J. Kocienski, in“Protecting Groups”, Georg Thieme Verlag Stuttgart, New York 1994 and,Theodora W. Greene and Peter G. M. Wuts in “Protective Groups in OrganicSynthesis”, Wiley-Interscience, 1991.

[0091] Compounds of this invention can be isolated in association withsolvent molecules by crystallization from evaporation of an appropriatesolvent. The pharmaceutically acceptable acid addition salts of thecompounds of formula (I), which contain a basic center, may be preparedin a conventional manner. For example, a solution of the free base maybe treated with a suitable acid, either neat or in a suitable solution,and the resulting salt isolated either by filtration or by evaporationunder vacuum of the reaction solvent. Pharmaceutically acceptable baseaddition salts may be obtained in an analogous manner by treating asolution of compound of formula (I) with a suitable base. Both types ofsalt may be formed or interconverted using ion-exchange resintechniques.

[0092] If the above set out general synthetic methods are not applicablefor the obtention of compounds of formula (I), suitable methods ofpreparation known by a person skilled in the art should be used.

[0093] When employed as pharmaceuticals, the pyrrolidine derivatives ofthe present invention are typically administered in the form of apharmaceutical composition. Hence, pharmaceutical compositionscomprising a compound of formula (I) and a pharmaceutically acceptablecarrier, diluent or excipient therefore are also within the scope of thepresent invention. A person skilled in the art is aware of a wholevariety of such carrier, diluent or excipient compounds suitable toformulate a pharmaceutical composition. Also, the present inventionprovides compounds for use as a medicament. In particular, the inventionprovides the compounds of formula (I) for use as antagonists of theoxytocin receptor, for the treatment or prevention of disorders mediatedby the oxytocin receptor in mammals, notably of humans, either alone orin combination with other medicaments, e.g. in combination with afurther OT antagonist.

[0094] The compounds of the invention, together with a conventionallyemployed adjuvant, carrier, diluent or excipient may be placed into theform of pharmaceutical compositions and unit dosages thereof, and insuch form may be employed as solids, such as tablets or filled capsules,or liquids such as solutions, suspensions, emulsions, elixirs, orcapsules filled with the same, all for oral use, or in the form ofsterile injectable solutions for parenteral (including subcutaneoususe). Such pharmaceutical compositions and unit dosage forms thereof maycomprise ingredients in conventional proportions, with or withoutadditional active compounds or principles, and such unit dosage formsmay contain any suitable effective amount of the active ingredientcommensurate with the intended daily dosage range to be employed.

[0095] When employed as pharmaceuticals, the pyrrolidine derivatives ofthis invention are typically administered in the form of apharmaceutical composition. Such compositions can be prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound. Generally, the compounds of this invention areadministered in a pharmaceutically effective amount. The amount of thecompound actually administered will typically be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

[0096] The pharmaceutical compositions of these inventions can beadministered by a variety of routes including oral, rectal, transdermal,subcutaneous, intravenous, intramuscular, and intranasal. Depending onthe intended route of delivery, the compounds are preferably formulatedas either injectable or oral compositions. The compositions for oraladministration can take the form of bulk liquid solutions orsuspensions, or bulk powders. More commonly, however, the compositionsare presented in unit dosage forms to facilitate accurate dosing. Theterm “unit dosage forms” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient. Typical unit dosage forms include prefilled,premeasured ampoules or syringes of the liquid compositions or pills,tablets, capsules or the like in the case of solid compositions. In suchcompositions, the pyrrolidine compound is usually a minor component(from about 0.1 to about 50% by weight or preferably from about 1 toabout 40% by weight) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

[0097] Liquid forms suitable for oral administration may include asuitable aqueous or nonaqueous vehicle with buffers, suspending anddispensing agents, colorants, flavors and the like. Solid forms mayinclude, for example, any of the following ingredients, or compounds ofa similar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatine; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavoring agent such as peppermint, methyl salicylate, or orangeflavoring.

[0098] Injectable compositions are typically based upon injectablesterile saline or phosphate-buffered saline or other injectable carriersknown in the art. As above mentioned, the pyrrolidine derivatives offormula (I) in such compositions is typically a minor component,frequently ranging between 0.05 to 10% by weight with the remainderbeing the injectable carrier and the like.

[0099] The above described components for orally administered orinjectable compositions are merely representative. Further materials aswell as processing techniques and the like are set out in Part 8 ofRemington's Pharmaceutical Sciences, 17th Edition, 1985, MarckPublishing Company, Easton, Pa., which is incorporated herein bereference.

[0100] The compounds of this invention can also be administered insustained release forms or from sustained release drug delivery systems.A description of representative sustained release materials can also befound in the incorporated materials in Remington's PharmaceuticalSciences.

[0101] In the following the present invention shall be illustrated bymeans of some examples which are not construed to be viewed as limitingthe scope of the invention. The HPLC, NMR and MS data provided in theexamples described below were obtained as followed. The followingabbreviations are hereinafter used in the accompanying examples: min(minute), hr (hour), g (gram), mmol (millimole), m.p. (melting point),eq (equivalents), mL (milliliter), μL (microliters), mL (milliliters),ACN (Acetonitrile), DBU (Diazabicyclo [5.4.0]undec-7-ene), DIEA(Diisopropylethylamine), CDCl₃ (deuterated chloroform), cHex(Cyclo-hexanes), DCM (Dichloromethane), DECP (Diethylcyanophosphonate),DIC (Diisopropyl carbodiimide), DMAP (4-Dimethylaminopyridine) DMF(Dimethylformamide), DMSO (Dimethylsulfoxide), DMSO-d₆ (deuterateddimethylsul-foxide), EDC(1-(3-Dimethyl-amino-propyl)-3-ethylcarbodiimide), EtOAc (Ethylacetate), Et₂O (Diethyl ether), HOBt (1-Hydroxybenzotriazole), K₂CO₃(potassium carbonate), NaH (Sodium hydride), NaHCO₃ (Sodiumbicarbonate), nBuLi (n Butyllithium), TBTU(O-Benzotriazolyl-N,N,N′,N′-tetra-methyluronium-tetrafluoroborate), TEA(Triethylamine), TFA (Trifluoro-acetic acid), THF (Tetrahydrofuran),MgSO₄ (Magnesium sulfate), PetEther (Petroleum ether), rt (roomtemperature).

EXAMPLES

[0102] Intermediate 1:(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid

[0103] Commercial(2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxy-2-pyrrolidinecarboxylic acid(30 g, 0.13 mol) was dissolved in acetone (1500 ml). A mechanicalstirrer was placed in the flask and the solution stirred vigorously. Afreshly made solution of 8N chromic acid was prepared by dissolvingchromium trioxide (66.7 g, 0.667 mol) in water (40 ml), addingconcentrated sulphuric acid (53.3 ml) and adding enough water to bringthe solution volume to 115 ml. The 8N chromic acid solution (115 ml) wasthen added dropwise over a period of 30 minutes with continued vigorousstirring, the reaction's exotherm being maintained at the optimaltemperature of 25° C. by the use of an ice bath. After the completeaddition of the chromic acid, the reaction mixture was stirred for afurther 15 minutes—maintaining the optimal temperature of 25° C. Thereaction mixture was then quenched by the addition of methanol (20 ml).Exotherm controlled by the use of an ice bath and, if necessary, directaddition of a small amount of crushed ice to the reaction mixtureitself. The reaction mixture was filtered through a Celite pad and thenconcentrated in vacuo. The resulting acidic solution was then extractedwith ethyl acetate (3×300 ml) and the combined organic layers washedwith brine (2×100 ml). Organics then dried with magnesium sulfate andconcentrated in vacuo. Crude product recrystallised from ethyl acetateto give the white crystalline product,(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid (22.55g, 76%). The antipodal intermediate,(2R)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid, wasmade according to the same protocol, starting from commercial(2R,4S)-1-(tert-butoxycarbonyl)-4-hydroxy-2-pyrrolidinecarboxylic acid.

[0104] 1H NMR (360 MHz, CDCl3): 1.4 (m, 9H), 2.5-3.0 (m, 2H), 3.7-3.9(m, 2H), 4.75 (dd, 1H)

[0105] Intermediate 2: 1-tert-butyl 2-methyl(2S-4-oxo-1,2-pyrrolidinedicarboxylate

[0106] A solution of(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid (1 g,4.3 mmol) in a 1:1 mixture of methanol and toluene (60 ml) was made.Trimethylsilyl diazomethane (6.5 ml of a 2M solution in hexanes, 13mmol) was then added dropwise to the stirred solution at roomtemperature under nitrogen. After completion of the evolution ofnitrogen gas, the resulting yellow solution was evaporated in vacuo, andthe residue filtered through a pad of silica gel, eluting with ethylacetate. Removal of solvent from the filtrate gave a yellow oil (1.05 g,near quantitative yield).

[0107]¹H NMR (400 MHz, CDCl₃): 1.4 (m, 9H), 2.5 (m, 1H), 2.8-2.9 (m, 1H)3.7 (s, 3H), 3.9 (m, 2H), 4.6-4.8 (m, 1H).

[0108] Intermediate 3: 1-tert-butyl 2-methyl(2S,4EZ)-4-(chloromethylene)-1,2-pyrrolidinedicarboxylate

[0109] Chloromethyltriphenylphosphonium iodide (270 mg, 0.62 mmol) wasadded to a solution of potassium tert-butoxide (67 mg, 0.59 mmol) inanhydrous diethyl ether (5 ml) under nitrogen and the resulting brightyellow mixture stirred for 30 minutes at ambient temperature. Thereaction was then cooled to 0° C. and a solution of 1-tert-butyl2-methyl (2S)-4-oxo-1,2-pyrrolidinedicarboxylate (100 mg, 0.41 mmol in 2ml anhydrous diethyl-ether) was added dropwise. The reaction was thenwarmed to room temperature and stirred for 30 minutes before addingsaturated aqueous ammonium chloride solution (0.5 ml). The organic layerwas removed in vacuo, and the aqueous washed with diethyl ether (3×5ml). The combined organic layers were dried with brine and magnesiumsulfate before filtering and removal of solvent. The desired product wasisolated by silica gel chromatography, eluting with 15% ethyl acetate inhexanes to give 105 mg (93% yield) as a off-white wax.

[0110]¹H NMR (400 MHz, CDCl₃): 1.4 (9H, m), 2.6-2.75 (m, 1H), 2.8-3.0(m, 1H), 3.65 (s, 3H), 4.1 (m, 2H), 4.44.5 (m, 1H)5.9-6.0 (m, 1H).

[0111] Intermediate 4: 1-tert-butyl 2-methyl(2S)-4-methylene-1,2-pyrrolidinedicarboxylate

[0112] Methyltriphenylphosphonium bromide (22 g, 61.6 mmol) was added toa solution of potassium tert-butoxide (6.5 g, 57.6 mmol) in anhydrousdiethyl ether (450 ml) at 0° C. under nitrogen and the resulting brightyellow mixture stirred for 30 minutes. A solution of 1-tert-butyl2-methyl (2S)-4-oxo-1,2-pyrrolidinedicarboxylate (10 g, 41.1 mmol in 150ml anhydrous diethyl ether) was added slowly to the reaction mixture,which was then warmed at 35° C. for 3 h. Saturated aqueous ammoniumchloride solution (0.5 ml) was then added. The organic layer wasremoved, and the aqueous washed with diethyl ether (3×5 ml). Thecombined organic layers were dried with brine and magnesium sulfatebefore filtering and removal of solvent. Silica gel chromatography,eluting with 15% ethyl acetate in hexanes gave the desired pro-duct 6.9g (70% yield) as a off-white wax.

[0113]¹H NMR (400 MHz, CDCl₃): 1.4 (9H, m), 2.5 (m, 1H), 2.8 (m, 1H),3.65 (s, 3H), 4.0 (m, 2H), 4.3-4.5 (m, 1H), 4.9 (m, 2H).

[0114] Intermediate 5: 1-tert-butyl 2-methyl(2S,4EZ)-4-(cyanomethylene)-1,2-pyrrolidinedicarboxylate

[0115] Diethyl cyanomethyl phosphonate (0.86 ml, 4.4 mmol) was dissolvedin dry THF (50 ml) and the solution cooled to 0° C. Sodium hydride (205mg of a 60% suspension in parrafin oil, 5.1 mmol) was then addedcautiously and the reaction stirred for 30 min. The reaction mixture wasthen cooled to −78° C. and a solution of 1-tert-butyl 2-methyl(2S)-4-oxo-1,2-pyrrolidinedicarboxylate (1.0 g, 4.1 mmol) in dry THF (5ml) was added dropwise. The reaction was then allowed to reach roomtemperature. Saturated aqueous ammonium chloride solution (5 ml) wasthen added, followed by ethyl acetate (100 ml). (The organic layer wasremoved, and the aqueous washed with ethyl acetate (3×5 ml). Thecombined organic layers were dried with brine and magnesium sulfatebefore filtering and removal of solvent. Silica gel chromatography,eluting with 35% ethyl acetate in hexanes gave the desired compound (860mg, 80%) as an off-white wax.

[0116]¹H NMR (360 MHz, CDCl₃): 1.4 (m, 9H), 2.7-3.0 (m, 1H), 3.1-3.3 (m,1H), 3.7 (m, 3H), 4.24.4 (m, 2H), 4.54.7 (m, 1H), 5.4 (m, 1H).

[0117] Intermediate 6: 1-tert-butyl 2-methyl(2S,4EZ)-4-benzylidene-1,2-pyrrolidinedicarboxylate

[0118] Potassium-tert-butoxide (6.1 g, 54 mmol) was added portionwise toa solution of benzyltriphenylphosphonium chloride (22.45 g, 58 mmol) inanhydrous dichloromethane (400 ml) and the reaction stirred at ambienttemperature for 1 h. The solution was then cooled to 0° C. and asolution of 1-tert-butyl 2-methyl(2S)-4-oxo-1,2-pyrrolidinedicarboxylate (9.36 g, 38.5 mmol) in drydichloromethane (30 ml) was added dropwise. After stirring for a further1 h at 0° C. the reaction was stirred for a further 3 h at ambienttemperature. Saturated aqueous ammonium chloride solution (30 ml) wasthen added. The organic layer was removed, and the aqueous washed withdichloromethane (3×20 ml). The combined organic layers were dried withbrine and magnesium sulfate before filtering and removal of solvent.Silica gel chromato-graphy, eluting with 30% ether in hexanes gave thedesired product 8.65 g (71% yield) as a pale yellow wax.

[0119]¹H NMR (400 MHz, CDCl₃): 1.5 (m, 9H), 2.8-3.0 (m, 1H), 3.2 (m,1H), 3.7 (m, 3H), 4.24.4 (m, 2H), 4.5-4.6 (m, 1H), 6.3-6.4 (m, 1H),7.1-7.5 (m, 5H).

[0120] Intermediate 7:(2S,4EZ)-1-(tert-butoxycarbonyl-4-(methoxyimino)-2-Pyrrolidinecarboxylicacid

[0121] A solution was made containing(2w)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid (5.0 g,21 mmol) and O-methylhydroxylamine hydrochloride (2.7 g, 32.8 mmol) inchloroform (100 ml) containing triethyl-amine (5.5 g, 55 mmol). Thereaction mixture was then stirred at ambient temperature over-night,prior to removal of solvent. The resultant crude reaction mixture wasdissolved in ethyl acetate (I 50 ml) and washed rapidly with 1N HCl (40ml). The acidic layer was then extracted with ethyl acetate (3×20 ml)and the combined organic layers washed with brine before drying overmagnesiom sulfate, filtering and removal of solvent in vacuo. Thedesired product (5.3 g, 94%) was isolated as a pale yellow oil.

[0122]¹H NMR (400 MHz, CDCl₃): 1.45 (m, 9H), 2.8-3.2 (m, 2H), 3.9 (s,3H), 4.2 (m, 2H), 4.54.7 (m, 1H).

[0123] Intermediate 8: (2S,4EZ)-1-(tert-butoxycarbonyl-4-(ethoxyimino)-2-pyrrolidinecarboxylicacid

[0124] A solution was made containing(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid (5.0 g,22 mmol) and O-ethylhydroxylamine hydrochloride (6.4 g, 65.5 mmol) in a1:1 mixture of pyridine and ethanol (100 ml). The reaction was heated toreflux for 2.5 h before cooling and removal of solvent. The residue wasdissolved in ethyl acetate and washed rapidly with 1.3N HCl (40 ml). Theacidic layer was then extracted with ethyl acetate (3×20 ml) and thecombined organic layers washed with brine before drying over magnesiomsulfate, filtering and removal of solvent in vacuo. The desired product(5.5 g, 93%) was isolated as a pale yellow oil.

[0125]¹HNMR (400 MHz, DMSO): 1.3 (t, 3H), 1.55 (m, 9H), 2.9-2.7 (m, 1H),3.4-3.1 (m, 1H), 4.1-4.3 (m, 4H), 4.6 (m, 1H), 12-13.5 (br, 1H).

[0126] Intermediate 9:(2S,4EZ)-4-[(allyloxy)imino]-1-(tert-butoxycarbonyl)-2-pyrrolidinecarboxylicacid

[0127] A solution was made containing(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid (5.0 g,22 mmol) and O-allylhydroxylamine hydrochloride monohydrate (7.2 g, 65.5mmol) in a 1:1 mixture of pyridine and ethanol (100 ml). The reactionwas heated to reflux for 2.5 h before cooling and removal of solvent.The residue was dissolved in ethyl acetate and washed rapidly with 1.3NHCl (40 ml). The acidic layer was then extracted with ethyl acetate(3×20 ml) and the combined organic layers washed with brine beforedrying over magnesium sulfate, filtering and re-moval of solvent invacuo. The desired product (5.9 g, 94%) was isolated as a pale yellowoil.

[0128]¹H NMR (400 MHz, CDCl₃): 1.5 (m, 9H), 2.8-3.2 (m, 2H), 4.2 (m,2H), 4.5-4.7 (m, 3H), 5.25 (m, 2H), 5.9 (m, 1H), 11.1 (broad S, 1H).

[0129] Intermediate 10: 1-[(aminooxy)methyl]-4-methoxy-benzene

[0130] A solution was made of Boc hydroxylamine (2.0 g, 17.1 mmol) indry THF (60 ml). Sodium hydride (1.1 g of a 60% suspension in paraffinoil, 25.7 mmol) was then added and the suspension stirred. A catalyticamount of KI was then added to the reaction prior to the cautiousaddition of 4-methoxybenzyl chloride (3.2 g, 20.4 mmol). The reactionwas then allowed to stir overnight before removal of solvent in vacuo.The residue was taken up with diethyl ether (100 ml) and HCl gas bubbledin for 20 minutes, causing the start of precipitation of the product.The flask was stoppered and left to stand overnight. The product wasthen filtered off as a off-white wax (39-52% yield according to varyingbatches).

[0131]¹H NMR (400 MHz, D₂O):3.8 (s, 3H), 5 (s, 2H), 7.0 (d, 2H), 7.4 (d,2H).

[0132] Intermediate 11:(2S,4EZ)-1-(tert-butoxycarbonyl-4-{[(4-methoxybenzyl)oxy]imino}-2-pyrrolidine-carboxylicacid

[0133] The same method as employed in the preparation of Intermediate 7,but starting from(2S)-1-(tert-butoxycarbonyl)-4-oxo-2-pyrrolidinecarboxylic acid(Intermediate 1) and 1[(aminooxy)methyl]-4-methoxy-benzene (Intermediate10) gave the title compound as a gum in a 85% yield.

[0134]¹H NMR (400 MHz, DMSO): 1.5 (m, 9H), 2.7-2.9 (m, 1H) 3.9 (s, 3H),4.2 (m, 3H), 4.6 (m, 1H), 5.15 (s, 2H), 7.1 (d, 2H), 7.45 (d, 2H).

[0135] Intermediate 12: 2′-methyl[1,1′-biphenyl]-4-carboxylic acid

[0136] To a mixture of 4-bromobenzoic acid (30 g, 0.15 mol),2-methylphenylboronic acid (24 g, 0.15 mol), sodium carbonate (250 g) intoluene (500 mL) and water (500 mL) was addedtetrakis-triphenylphosphine palladium(0) (9 g, 0.0074 mol) undernitrogen atmosphere. The reaction mixture was refluxed for 10 h. Afterthis time, 100 ml of 10% NaOH were added to the reaction mixture, theaqueous layer was separated and washed with toluene (2×260 mL).Acidification of the aqueous layer with 3N HCl solution gave a solidproduct, which was filtered, washed with water and dried. The crudeproduct was then crystallised from toluene to yield2′-methyl[1,1′-biphenyl]-4-carboxylic acid (20 g, 62.5%).

[0137] Conversely, the product could also be obtained from1-bromo-2-methylbenzene and 4-carboxybenzeneboronic acid, usinganalogous conditions.

[0138]¹H NMR (300 MHz, DMSO): 2.2 (s, 3H), 7.2-7.4 (m, 4H), 7.43 (d, J=9Hz, 2H), 7.99 (d, J=9 Hz, 2H), 13 (b, 1H).

[0139] Similarly, using the appropriate commercial boronic acids andarylbromides, the following, related intermediate 1,1′-biphenylderivatives (12) were obtained: 4′-methyl[1,1′-biphenyl]-4-carboxylicacid; 2′,3-dimethyl[1,1′-biphenyl]-4-carboxylic acid;2′,6′-dimethyl[1,1′-biphenyl]-4-carboxylic acid;2-methyl[1,1′-biphenyl]4-carboxylic acid;3-methyl[1,1′-biphenyl]-4-carboxylic acid;2,2′-dimethyl[1,1′-biphenyl]-4-carboxylic acid;2′-methoxy[1,1′-biphenyl]-4-carboxylic acid;3′-methoxy[1,1′-biphenyl]-4-carboxylic acid;4′-methoxy[1,1′-biphenyl]4-carboxylic acid;2′-chloro[1,1′-biphenyl]4-carboxylic acid;3′-chloro[1,1′-biphenyl]-4-carboxylic acid;4′-chloro[1,1′-biphenyl]4-carboxylic acid;3′,4′-dichloro[1,1′-biphenyl]4-carboxylic acid;2′-(trifluoromethyl)[1,1′-biphenyl]-4-carboxylic acid;3′-(trifluoromethyl)[1,1′-biphenyl]-4-carboxylic acid;2′-cyano[1,1′-biphenyl]-4-carboxylic acid;2′,4′-difluoro[1,1′-biphenyl]4-carboxylic acid; 4-(2-pyridinyl)benzoicacid; 4-(3-pyridinyl)benzoic acid; 4-(4-pyridinyl)benzoic acid;4-(5-pyrimidinyl)benzoic acid; and others.

[0140] Intermediate 13: 4-(3-methyl-2-pyridinyl)benzoic acid

[0141] A mixture of 2-bromo-3-methylpyridine (22.5 g, 0.1312 mol),4(hydroxymethyl)phenylboronic acid (25 g, 0.164 mol), Pd(PPh₃)₄ (9.5 g,0.0082 mol), and sodium carbonate (200 g in 500 ml of water) in toluene(750 ml) were refluxed under nitrogen atmosphere for 15 h. Separated thetoluene layer and distilled under reduced pressure to give a residue.The residue was then purified by column chromatography to yield[4-(3-methyl-2-pyridinyl)phenyl]methanol (12 g, 47%).

[0142] To a solution of [4-(3-methyl-2-pyridinyl)phenyl]methanol (12 g,0.06 mol) in dry DMF (150 mL) was added pyridiniumdichromate (91 g, 0.24mol) and stirred at RT for 3 days. The reaction mixture was poured intowater and extracted with ethyl acetate (250 mL). The organic layer waswashed with water, brine, dried and concentrated. The crude was purifiedby column chromatography over silica gel to give4-(3-methyl-2-pyridinyl)benzoic acid (3 g, 25%) as white solid.

[0143]¹H NMR (300 MHz, DMSO): 2.3 (s, 3H), 7.33 (dd, J=7.5 Hz, 5 Hz,1H), 7.67 (d, J=8 Hz, 2H), 7.75 (d, J=7.5 Hz, 1H), 8.01 (d, J=8 Hz, 2H),8.50 (d, J=5 Hz, 1H), 13 (b, 1H).

[0144] Intermediate 14: 4-(1-oxido-3-pyridinyl)benzoic acid

[0145] To a mixture of 4-tolylboronic acid (38 g, 0.28 mol),3-bromopyridine (44 g, 0.28 mol), Na₂CO₃ (200 g) in toluene (500 ml) andwater (500 ml) was added Pd(PPh₃)₄ (16 g, 0.014 mol), and refluxed for16 h. The reaction mixture was cooled, and the separated organic layerwas washed with water and brine, and dried. The solvent was removed togive 4-(3-pyridyl)toluene (42 g, 90%).

[0146] To a mixture of 4-(3-pyridyl)toluene (35 g, 0.207 mol) inpyridine (400 ml) and water (400 ml) was added KMnO₄ (163 g, 1.03 mol)in portions and refluxed for 12 h. The reaction mixture was filteredthrough celite and acidified with conc. HCl. The product was washed withwater and dried to give 4-(3-pyridyl)benzoic acid (32 g, 76%) as a whitesolid. To a mixture of 4-(3-pyridyl)benzoic acid (22 g, 0.11 mol) in THF(2.51), mCPBA (152 g, 0.44 mol, 50%) was added and stirred at RT for 12h. The solid was filtered, and washed with THF to give4-(1-oxido-3-pyridinyl)benzoic acid (20 g, 86%).

[0147]¹H NMR (300 MHz, DMSO): 7.5-7.8 (m, 5H), 7.9 (d, J=8 Hz, 2H), 8.33(d, J=5 Hz, 2H).

[0148] Similarly, starting from 4-tolylboronic acid (45 g, 0.33 mol) and2-bromopyridine (52 g, 0.33 mol), the related intermediate4-(1-oxido-2-pyridinyl)benzoic acid was obtained.

Example 1 General Procedure for the Saponification of methylesters ofoximether- and/or olefin-type 2-pyrrolidinecarboxylic acid intermediates(Schemes 3, 7):

[0149] A solution of sodium hydroxide (73 mg, 1.81 mmol) in water (1.2ml) was added to a proline oximether methyl ester derivative, e.g.methyl (2 S,4EZ)-4-(methoxyimino)-1-[(2′methyl[1,1′-biphenyl]4-yl)carbonyl]-2-pyrrolidinecarboxylate(391 mg, 1.1 mmol) in 3:1 dioxane:water (12 ml) and the reaction stirredfor 3 h. The reaction mixture was then washed with diethyl ether (2×10ml), and the aqueous phase acidified to pH 2 (0.1N HCl) and extractedinto ethyl acetate. The ethyl acetate layer was then dried overmagnesium sulfate, filtered and the solvent was then removed in vacuo togive the desired product, e.g.(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylicacid in 91% yield as an oil which was used without further purification.

[0150]¹H NMR (300 MHz, CDCl₃): 2.25 (m, 3H, ArCH₃), 2.96-3.35 (m, 2H),3.84 (m, 3H), 4.37 (br s, 2H), 5.17 (m, 1H), 7.14-7.32 (m, 4H, H arom.),7.34-7.44 (m, 2H, H arom.), 7.53-7.63 (m, 2H, H arom.). M⁺(APCI⁺): 353:M⁻(APCI⁻): 351.1.

Example 2 General Protocols for the Esterification of oximether- and/orolefin-type 2-pyrrolidinecarboxylic acid intermediates (Schemes 2, 5,7):

[0151] a) Methylesters (e.g. 1-tert-butyl 2-methyl(2S,4EZ-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate):

[0152] A solution of the oximether- and/or olefin-type2-pyrrolidinecarboxylic acid intermediate, e.g.(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid (0.648 g, 2.5 mmol), in a 1:1 mixture of methanol and toluene (35ml) was made. Trimethylsilyl diazomethane (3.8 ml of a 2M solution inhexanes, 7.5 mmol) was then added dropwise to the stirred solution atroom temperature under nitrogen. After completion of the evolution ofnitrogen gas, the resulting yellow solution was evaporated in vacuo, andthe residue filtered through a pad of silica gel, eluting with ethylacetate. Removal of solvent from the filtrate gave the methylesterproduct, e.g. 1-tert-butyl2-methyl(2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate, as ayellow oil (0.646 g, 95% yield).

[0153] b) Other esters (e.g. sec-butyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate):

[0154] A solution was made containing the the oximether- and/orolefin-type 2-pyrrolidinecarboxylic acid intermediate, e.g.(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylicacid (50 mg, 0.14 mmol), an alcohol, e.g. isobutanol (0.012 ml, 0.128mmol) and DMAP (6 mg, 0.05 mmol) in anhydrous DCM (5 ml). At 0° C., EDC(27 mg, 0.14 mmol) in DCM (2.5 mL) was added dropwise. The reactionmixture was stirred 2 h at 0° C. followed by 4 h at r.t.. The reactionmixture was concentrated in vacuo and the residue was redissolved inEtOAc. The resulting solution was washed with HCl 0.1N, water, NaHCO₃sat and brine and dried over magnesium sulfate. After filtration througha pad of silica and evaporation of the solvents, the desired product,e.g.(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylicacid was isolated as a mixture of two isomers as an oil in 69% yield(96.2% purity by HPLC).

[0155]¹H NMR (300 MHz, CDCl₃): 0.7-1.0 (m, 3H), 1.02-1.34 (m, 3H),1.38-1.72 (m, 2H), 2.24 (m, 3H, ArCH₃), 2.75-3.18 (m, 2H), 3.84 (m, 3H,NOCH₃), 4.12-4.48 (m, 2H), 4.54-5.18 (m, 2H), 7.13-7.29 (m, 4H),7.31-7.62 (m, 4H). M⁺(APCI⁺): 409.

[0156] Example 3: Cyclopentyl(2S,4EZ)-4-(methoxyimino-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate

[0157] Following the general methods as outlined in Example 2, startingfrom(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylicacid and cyclopentanol, the title compound was isolated, afterflash-chromatography, as a mixture of two isomers as an oil in 57% yield(95.6% purity by HPLC).

[0158]¹H NMR (300 MHz, CDCl₃): 1.47-1.98 (m, 8H), 2.24 (m, 3H, ArCH₃),2.73-3.14 (m, 2H), 3.84 (m, 3H, NOCH₃), 4.11-4.46 (m, 2H), 4.61 (br s,1H), 4.99-5.32 (m, 2H), 7.15-7.28 (m, 4H), 7.31-7.41 (m, 2H), 7.51-7.62(m, 2H). M⁺(APCI⁺): 421.

Example 4 General Protocol for the Solution-Phase Synthesis of OximetherPyrrolidine Derivatives of General Formula (I) (Scheme 1): e.g. methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[(1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate:methyl(2S,4E)-4-(methoximino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate;methyl(2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate;methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate.

[0159] a) Protocol for the N-Deprotection Step

[0160] Method A: A solution was made containing e.g. 1-tert-Butyl2-methyl (2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate (0.892g, 3.28 mmol), in anhydrous DCM (28 ml). TFA (20%, 7 mL) was addeddropwise. The mixture was stirred at r.t. for 20 min. Solvents wereevaporated and the desired product, e.g. methyl(2S,4EZ)-4-(methoxyimino)-2-pyrrolidinecarboxylate (0.564 g, quant.) wasisolated as a yellow oil and used without further purification.

[0161] Method B: A solution was made containing e.g. 1-tert-Butyl2-methyl (2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate (60 mg,0.22 mmol), in anhydrous DCM (6 ml). At 0° C., HCl gas was bubbledslowly through the reaction and the deprotection was followed by TLC.After approximately 30 minutes, the DCM was evaporated. The product wasconcentrated in vacuo from DCM (2-3 times) to remove the HCl. Thedesired product, e.g. methyl(2S,4EZ)-4-(methoxyimino)-2-pyrrolidinecarboxylate (38 mg, quant.) wasisolated as a yellow solid and used without further purification.

[0162] b) Protocol for the N-Capping Step

[0163] Method A (e.g. methyl(2S,4EZ)-4-4-(methoxyimino-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate):A solution of methyl-(2S,4EZ)-4-(methoxyimino)2-pyrrolidinecarboxylate(0.564 g, 3.28 mmol), 2′-methyl[1,1′-biphenyl]-4-carboxylic acid (0.765g, 3.60 mmol) and 4-dimethylaminopyridine (0.880 g, 7.21 mmol) in a 7:3mixture of DCM and DMF (30 ml) was made. EDC (0.691 mg, 3.60 mmol) wasadded slowly at 0° C. The reaction mixture was stirred overnight at r.t.It was washed with water (twice 20 ml), dried over MgSO₄, filtrated andevaporated in vacuo. The resulting crude product mixture, methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate,was purified by flash chromatography, unsing cyclohexane/EtOAc 8:2 aseluent. After several further chromatographies, (E)- and (Z)-isomerscould be separated: methyl(2S,4E)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]2-pyrrolidinecarboxylate(261 mg, 22%) was isolated as a colorless powder in 98.3% purity byHPLC, and methyl(2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate(237 mg, 20%) was isolated as a colorless powder in 98.3% purity byHPLC.

[0164] Methyl(2S,4E)₄-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]4-yl)carbonyl]-2-pyrrolidinecarboxylate:M.p. 38° C.; IR (neat) v 2952, 1743, 1640, 1405, 1206, 1177, 1045, 851cm⁻¹; ¹H NMR (300 MHz, CDCl₃): 2.27 (s, 3H, ArCH₃), 2.92-3.18 (m, 2H),3.81 (m, 3H), 3.87 (m, 3H), 4.37 (m, 2H), 5.20 (m, 1H), 7.16-7.32 (m,4H, H arom.), 7.35-7.42 (m, 2H, H arom.), 7.55-7.67 (m, 2H, H arom.).M⁺(APCI⁺): 367.3. Analysis calculated for C₂₈H₂₉N₃O₄ 0.1 H₂O: C, 68.50;H, 6.08; N, 7.61. Found: C, 68.23; H, 6.16; N, 7.45.

[0165] Methyl(2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate:M.p. 40° C.; IR (neat) v 2937, 1742, 1640, 1405, 1207, 1177, 1045, 754cm⁻¹; ¹HNMR (300 MHz, CDCl₃): 2.27(s, 3H, ArCH₃), 2.92-3.18 (m, 2H),3.81 (m, 3H), 3.87 (m, 3H), 4.37 (m, 2H), 5.20 (m, 1H), 7.16-7.32 (m,4H, H arom.), 7.35-7.42(m, 2H, H arom.), 7.55-7.67 (m, 2H, H arom.).M⁺(APCI⁺): 367.3. Analysis calculated for C₂₈H₂₉N₃O₄: C, 68.84; H, 6.05;N, 7.65. Found: C, 68.46; H, 6.26; N, 7.35.

[0166] Method B (e.g. methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate):To a solution of 2′-fluoro[1,1′-biphenyl]-4-carboxylic acid (69 mg, 0.32mmol.) in 9 ml THF, were added oxalyl chloride (0.09 mL, 0.99 mmol) andDMF (three drops) under ice cooling. The mixture was stirred for 2 h atrt. The solvent was removed affording the corresponding acyl chloride,2′-fluoro[1,1′biphenyl]-4-carbonyl chloride. The latter was nowdissolved in THF (7 mL) and added slowly on a 0° C. solution containingthe free NH-compound from the previous step, e.g. methyl(2S,4EZ)-4-(methoxyimino)-2-pyrrolidinecarboxylate (38 mg, 0.22 mmol),and triethylamine (2eq, 0.44 mmol, 0.06 ml) in THF/DCM 1:1 mixture (12ml). The reaction mixture was stirred overnight at r.t. Pol-trisaminewas added (69 mg, 3.45 mmol/g) in order to scavenge excess of acylchloride. The mixture was shaken 5 h, filtered and the resultingsolution was washed with NH₄Cl 20%, brine, and dried over MgSO₄. Afterfiltration and evaporation of the solvents, the resulting dark oil (3.26g) was purified by SPE (SAX sorbent) using neat DCM as eluent. Thedesired product, e.g. methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylatewas obtained as a mixture of two isomers as a white foam in 34% yield(97.3% purity by HPLC).

[0167] Methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylate:¹H NMR (300 MHz, CDCl₃): 2.80-3.20 (m, 2H), 3.70-3.85 (m, 6H), 4.07-4.40(m, 2H), 3.55-3.82 (m, 1H), 3.90-4.44 (m, 2H), 5.20 (m, 1H), 7.13-7.25(m, 2H), 7.30-7.46 (m, 2H), 7.61(m, 4H). M⁺(APCI^(+):) 371.2

[0168] c) E/Z-isomerisation

[0169] The pure E-isomer was isomerized to a mixture of the E/Z-isomersby the following procedure: the E-isomer was dissolved in dioxane/water3:1 mixture. NaOH (1.7 eq; 0.52 mL of NaOH 1.6N) was added and theresulting solution was stirred 2 h at r.t. The mixture was neutralysedwith HCl 0.1 N and lyophilised. The components of the resultingE/Z-mixture were separated and purified by flash chromatography usingsame conditions as described above.

Example 5 Methyl(2S,4EZ)-1-[(4′-fluoro[1,1′-biphenyl]4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0170] Following the general methods as outlined in Example 4, starting1-tert-butyl 2-methyl(2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate and4′-fluoro[1,1′-biphenyl]-4-carboxylic acid, the title compound wasisolated, after flash-chromatography, as a mixture of two isomers as anoil in 39% yield (97.6% purity by HPLC).

[0171]¹H NMR (300 MHz, CDCl₃): 2.72-3.20 (m, 2H), 3.74-3.87 (m, 6H),4.104.42 (m, 2H), 5.20 (m, 1H), 7.12-7.18 (m, 2H), 7.53-7.61 (m, 6H).M⁺(APCI⁺): 371.2

Example 6 Methyl(2S,4EZ)-4-(methoxyimino)-1-[4-(5-pyrimidinyl)benzoyl]-2-pyrrolidinecarboxylate

[0172] Following the general methods as outlined in Example 4, startingfrom I-tert-butyl 2-methyl(2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate and4-(5-pyrimidinyl)benzoic acid, the title compound was obtained, afterflash-chromatography, as a mixture of two isomers as an oil in 68% yield(93.0% purity by HPLC).

[0173]¹H NMR (300 MHz, CDCl₃): 2.64-3.20 (m, 2H), 3.74-3.87 (m, 6H),4.16-4.64 (m, 2H), 5.18 (m, 1H), 7.64-7.73 (m, 4H), 8.97 (d, 2H), 9.26(s, 1H). M⁺(APCI⁺): 355.3.

Example 7 Methyl(2S,4EZ)-1-([1,1′-biphenyl]-4-ylcarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0174] Following the general methods as outlined in Example 4, startingfrom 1-tert-butyl2-methyl(2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate and[1,1′-biphenyl]-4-carbonyl chloride, the title compound was isolated,after flash-chromatography, as a mixture of two isomers as an oil in 31%yield (99% purity by HPLC).

[0175]¹H NMR (300 MHz, CDCl₃): 2.88 (m, 1H), 3.07 (m, 1H), 3.80 (m, 6H),4.204.45 (m, 2H), 4.65 (br s, 1H), 5.15 (m, 1H), 7.33-7.49 (m, 4H),7.54-7.69 (m, 5H). M⁺(APCI⁺): 353.2.

Example 8 Methyl(2S,4EZ)-4-(methoxyimino)-1-[4-(2-pyridinyl)benzoyl]-2-pyrrolidinecarboxylate

[0176] Following the general method as outlined in Example 4 (Method B),starting from 1-tert-butyl 2-methyl(2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate and4-(2-pyridinyl)benzoic acid, the title compound was obtained, afterflash-chromatography (eluent cyclohexane/ethyl acetate 8:2), as amixture of two isomers in 45% yield (95% purity by HPLC).

[0177]¹H NMR (300 MHz, CDCl₃): 2.90-3.20 (m, 2H), 3.70-3.85 (m, 6H),4.26-4.35 (m, 2H), 5.15 (m, 1H), 7.26 (m, 2H), 7.30-7.76 (m, 4H), 8.10(m, 2H), 8.73 (m, 1H). M⁺(ESI⁺): 354.

Example 9 Methyl(2S,4EZ)-4-(methoxyimino)-1-[4-(3-methyl-2-pyridinyl)benzoyl]-2-pyrrolidinecarboxylate

[0178] Following the general method as outlined in Example 4 (Method B),starting from 1-tert-butyl 2-methyl(2S,4EZ)-4-(methoxyimino)-1,2-pyrrolidinedicarboxylate and4-(3-methyl-2-pyridinyl)benzoic acid, the title compound was obtained,after flash-chromatography (eluent cyclohexane/ethyl acetate 8:2), as amixture of two isomers in 50% yield (100% purity by HPLC).

[0179]¹H NMR (300 MHz, CDCl₃); 2.38 (s, 3H), 2.80-3.20 (m, 2H),3.70-3.85 (m, 6H), 4.21-4.41 (m, 2H), 5.16 (m, 1H), 7.64 (m, 6H), 8.55(m, 1H). M⁺(APCI⁺): 368.2.

Example 10 General Protocol for the Solution-Phase Synthesis of Oxime orHydrazone Pyrrolidine Derivatives of General Formula (I) (Scheme 6)

[0180] a) Protocol for the Hydrolysis of the Oximether Group.

[0181] The starting oximether compounds, (0.14 mmol), paraformaldehydepowder (95%, 1.41 mmol) and Amberlyst 15 (30 mg) were mixed in acetonecontaining 10% of water (2 mL). The reaction was stirred 4 h at 60° C.Insoluble materials were filtered off and washed with a small amount ofacetone. The filtrate was concentrated and the residue was diluted withDCM (15 mL). The organic solution was washed with brine (10 mL), driedover Na2SO₄, and concentrated. The desired 4-ketopyrroldidine productwas isolated as a yellow oil and used without further purification(92%).

[0182] b) Protocol for the Formation of Oxime and/or Hydrazone Compounds

[0183] A solution was made containing the ketopyrrolidine derivativefrom the previous step (0.11 mmol) and hydroxylamine hydrochloride (0.17mmol) in chloroform (1 ml) containing triethylamine (0.29 mmol), orhydrazine hydrate (4% in EtOH). The reaction mixtures were then stirredat ambient temperature for one day, prior to removal of solvent. Theresultant crude reaction mixtures were purified by column chromatographyusing DCM/MeOH (25:1) to collect the desired oxime or hydrazoneproducts, respectively.

Example 11 General Protocol for the Solid-Phase Synthesis of PyrrolidineEster Derivatives of General Formula (I) (Scheme 8):

[0184] a) Loading Step

[0185] Kaiser oxime resin (16.5 g, loading 1.57 mmol/g) was added to asolution of the relevant pyrrolidine carboxylic acid building block(51.8 mmol) and diisopropylcarbodiimide (8.1 ml, 51.8 mmol) in drydichloromethane (150 ml). The resulting suspension was shaken overnightbefore filtering at the pump and washing sequentially with DMF, DCM andfinally diethyl ether before drying at room temperature in vacuo.

[0186] b) N-Deprotection Step

[0187] The resin obtained in the loading step was shaken with a 20%solution of trifluoroacetic acid in dichloromethane (200 ml) for 30minutes prior to filtering at the pump and washing sequentially withaliquots of DMF, DCM and finally diethyl ether before drying at roomtemperature in vacuo.

[0188] c) N-Capping Step

[0189] The resin from the previous step was transferred into a 96-wellfilter-plate (approx. 50 mg of dry resin/well) and each well treatedwith an N-reactive derivatising agent, e.g. with either of the followingsolutions:

[0190] a) an acid chloride (0.165 mmol) and diisopropylethylamine (0.165mmol) in dry dichloromethane (1 ml), overnight

[0191] b) an acid (0.165 mmol) and DIC (0.165 mmol) in, depending on thesolubility of the carboxylic acid, dry dichloromethane or NMP (1 ml)overnight.

[0192] The tubes were closed with a stopper and shaken overnight atambient temperature. The resins were then filtered, washing the resinsequentially with aliquots of DMF, DCM and finally diethyl ether beforedrying at room temperature in vacuo.

[0193] d) Cleavage Step

[0194] A solution of MeOH (20 eq, 50 μL) and TEA (1 eq. 8 μL) in DCM(1.45 mL) was added to each tube containing the resin from the previousstep. They were shaken for 2 days at room temperature. They were thenfiltered into individual vials and the solvent removed in a vacuumcentrifuge to yield about 10 mg of the corresponding products (between40 and 50% yield). The products were characterised by LC (MaxPlotdetection between 230 and 400 nm) and mass spec-trometry (ES+). All ofthe following examples were identified based on the observation of thecorrect molecular ion in the mass spectrum, and were shown to be atleast 40% pure (usually 60-95% pure) by LC.

Example 12 Ethyl(2S,4EZ)-1-([1,1′-biphenyl]-4-ylcarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0195] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, [1,1′-biphenyl]-4-carbonyl chloride, and ethanol, the titlecompound was isolated as a mixture of two isomers in 87.2% purity byHPLC.

[0196]¹H NMR (300 MHz, CDCl₃): 1.43 (m, 314), 2.76-3.18 (m, 2H),3.74-3.94 (m, 5H), 4.094.48 (m, 2H), 5.14 (m, 1H), 7.32-7.51 (m, 4H),7.52-7.70 (m, 5H). M⁺(APCI⁺): 367.

Example 13 Methyl(2S,4EZ)-1-[(2′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0197] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 2′-chloro[1,1′-biphenyl]-4-carboxylic acid, and methanol, thetitle compound isolated as a mixture of two isomers in 92.3% purity byHPLC.

[0198]¹H NMR (300 MHz, CDCl₃): 2.85 (m, 1H), 3.05 (m, 1H), 3.72-3.94 (m,6H), 4.27 (m, 1H), 4.41 (m, 1H), 4.68 (br s, 1H), 5.15 (m, 1H),7.23-7.35 (m, 3H), 7.42-7.53 (m, 3H), 7.557.64 (m, 2H). M⁺(APCI⁺): 387.

Example 14 Methyl(2S,4EZ)-1-[(2′-cyano[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0199] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 2′-cyano[1,1′-biphenyl]-4-carboxylic acid, and methanol, the titlecompound was isolated as a mixture of two isomers in 91.6% purity byHPLC.

[0200]¹H NMR (300 MHz, CDCl₃): 2.75-3.18 (m, 2H), 3.72-3.94 (m, 6H),4.25 (m, 1H), 4.41 (m, 1H), 4.62 (br s, 1H), 5.15 (m, 1H), 7.40-7.55 (m,3H),7.56-7.71 (m, 4H), 7.77 (m, 1H). M⁺(APCI⁺): 378.

Example 15 Methyl(2S,4EZ)-4-(methoxyimino)-1-{[2′-(trifluoromethyl)[1,1′-biphenyl]-4-yl]carbonyl}-2-pyrrolidinecarboxylate

[0201] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 2′-(trifluoromethyl)[1,1′-biphenyl]-4-carboxylic acid, andmethanol, the title compound was isolated as a mixture of two isomers in86.3% purity by HPLC.

[0202]¹H NMR (300 MHz, CDCl₃): 2.85 (m, 1H), 3.06 (m, 1H), 3.72-3.94 (m,6H), 4.26 (m, 1H), 4.41 (m, 1H), 4.62 (br s, 1H), 5.17 (m, 1H), 7.29 (m,1), 7.37 (m, 2H), 7.48 (m, 1H), 7.56 (m, 3H), 7.74 (m, 1H). M⁺(APCI⁺):421.

Example 16 Methyl(2S,4EZ)-1-[(2′-methoxy[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0203] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 2′-methoxy[1,1′-biphenyl]-4-carboxylic-acid, and methanol, thetitle compound was isolated as a mixture of two isomers in 92.1% purityby HPLC.

[0204]¹H NMR (300 MHz, CDCl₃): 2.85 (m, 1H), 3.06 (m, 1H), 3.67-3.94 (m,9H), 4.234.49 (m, 1H), 5.14 (m, 1H), 6.94-7.07 (m, 2H), 7.26-7.67 (m,6H). M⁺(APCI⁺): 383.

Example 17 Methyl(2S,4EZ)-1-[(2′,6′-dimethyl[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0205] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 2′,6′-dimethyl[1,1′-biphenyl]-4-carboxylic acid, and methanol, thetitle compound was isolated as a mixture of two isomers in 88.3% purityby HPLC.

[0206]¹H NMR (300 MHz, CDCl₃): 1.98 (s, 3H), 2.00 (s, 3H), 2.67-3.18 (m,2H), 3.65-3.94 (m, 6H), 4.12-4.75 (m, 3H), 5.15 (m, 1H), 7.05-7.27 (m,5H), 7.35-7.67 (m, 2H). M⁺(APCI⁺): 381.

Example 18 Methyl(2S,4EZ)-1-[(2′,3-dimethyl[1,1′-biphenyl]4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0207] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 2′,3-dimethyl[1,1′-biphenyl]-4-carboxylic acid, and methanol, thetitle compound was isolated as a mixture of two isomers in 81.4% purityby HPLC.

[0208]¹H NMR (300 MHz, CDCl₃): 2.20-2.45 (m, 6H), 2.72-3.20 (m, 2H),3.65-3.94 (m, 6H), 3.96-4.24 (m, 2H), 5.11 (m, 1H), 7.10-7.29 (m, 7H).M⁺(APCI⁺): 381.

Example 19 Methyl(2S,4EZ)-1-[(3-methyl[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0209] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 3-methyl[1,1′-biphenyl]4-carboxylic acid, and methanol, the titlecompound isolated as a mixture of two isomers in 82.3% purity by HPLC.

[0210]¹H NMR (300 MHz, CDCl₃):2.40 (m, 3H, ArCH₃), 2.65-3.20 (m, 2H),3.74-3.87 (m, 6H), 3.92-4.20 (m, 2H), 5.10 (m, 1H), 7.16-7.48 (m, 2H),7.56 (m, 2H). M⁺(APCI⁺): 367.

Example 20 Methyl(2S,4EZ)-1-[(3′,4′-dichloro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylate

[0211] Following the general method as outlined in Example 11, startingfrom(2S,4EZ)-1-(tert-butoxycarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylicacid, 3′,4′-dichloro[1,1′-biphenyl]-4-carboxylic acid, and methanol, thetitle compound was isolated as a mixture of two isomers in 91.9% purityby HPLC.

[0212]¹H NMR (300 MHz, CDCl₃): 2.72-3.18 (m, 2H), 3.65-3.94 (m, 6H),4.21 (m, 1H),4.37 (m, 1H), 5.15 (m, 1H), 7.35-7.69 (m, 7H). M⁺(APCI⁺):421.

Example 21 Preparation of a Pharmaceutical Formulation

[0213] The following formulation examples illustrate representativepharmaceutical compositions according to the present invention, beingnot restricted thereto.

[0214] Formulation 1—Tablets

[0215] A pyrrolidine compound of formula (1) is admixed as a dry powderwith a dry gelatin binder in an approximate 1:2 weight ration. A minoramount of magnesium stearate is added as a lubricant. The mixture isformed into 240-270 mg tablets (80-90 mg of active pyrrolidine compoundper tablet) in a tablet press.

[0216] Formulation 2—Capsules

[0217] A pyrrolidine compound of formula (1) is admixed as a dry powderwith a starch diluent in an approximate 1:1 weight ratio. The mixture isfilled into 250 mg capsules (125 mg of active pyrrolidine compound percapsule).

[0218] Formulation 3—Liquid

[0219] A pyrrolidine compound of formula (I) (1250 mg), sucrose (1.75 g)and xanthan gum (4 mg) are blended, passed through a No. 10 mesh U.S.sieve, and then mixed with a previously prepared solution ofmicrocrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50mg) in water. Sodium benzoate (10 mg), flavor, and color are dilutedwith water and added with stirring. Sufficient water is then added toproduce a total volume of 5 mL.

[0220] Formulation 4—Tablets

[0221] A pyrrolidine compound of formula (I) is admixed as a dry powderwith a dry gelatin binder in an approximate 1:2 weight ratio. A minoramount of magnesium stearate is added as a lubricant. The mixture isformed into 450-900 mg tablets (150-300 mg of active pyrrolidinecompound) in a tablet press.

[0222] Formulation 5—Injection

[0223] A pyrrolidine compound of formula (1) is dissolved in a bufferedsterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/ml.

Example 22 Biological Assays

[0224] The compounds according to formula (I) may be subjected to thefollowing assays:

[0225] a) In Vitro Competition Binding Assay on hOT Receptor withScintillation Proximity Assay (see Cook, N. D. et al. PharmaceuticalManufacturing International 1992; p.49-53)

[0226] This assay allows to determine the affinity of the test compoundsfor the human Oxytocin (hOT) receptor. Membranes from HEK293EBNA (cellsexpressing the hOT receptor) were suspended in buffer containing 50 mMTris-HCl, pH 7.4, 5 mM MgCl2 and 0.1% BSA (w/v). The membranes (2-4 μg)were mixed with 0.1 mg SPA bead coated with wheat-germ aglutinin(WGA-PVT-Polyethylene Imine beads from Amersham) and 0.2 nM of theradiolabelled [¹²⁵I]-OVTA (OVTA being Omithin Vasoactive, an analogue ofOT for competitive binding experiments). Non-specific binding wasdetermined in the presence of 1 μM Oxytocin. The total assay volume was100 μl. The plates (Corning® NBS plate) were incubated at roomtemperature for 30 min and counted on a Mibrobeta® plate scintillationcounter. Competitive binding was performed in presence of compounds offormula (I) at the following concentrations: 30 μM, 10 μM, 1 μM, 300 nM,100 nM, 10 nM, 1 nM, 100 pM, 10 pM. The competitive binding data wereanalysed using the iterative, nonlinear, curve-fitting program, “Prism”(GraphPad Software, Inc).

[0227] The ability of the pyrrolidine derivatives of formula (I) toinhibit the binding of ¹²⁵I-OVTA to the OT-receptor was assessed usingthe above described in vitro biological assay. Representative values forsome example compounds are given in Table 1, where the binding affinityof the compounds is expressed by the IC₅₀ (μM) which is theconcentration upon which 50% inhibition of OT-R is achieved. From thesevalues, it can be derived that said test compounds according to formula(I) do show a significant binding to the oxytocin receptor.

[0228] According to a preferred embodiment, the compounds of theinvention display binding affinities (K_(i)(μM)) of less 0.40 μM, morepreferred of less than 0.1 μM. TABLE 1 Binding affinity human OT-RStructure IUPAC-Name IC₅₀ (μM)

Methyl (2S,4EZ)-1-([1,1′-biphenyl]-4- ylcarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylate 0.045

Methyl (2S,4EZ)-4-(methoxyimino)-i-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.028

Methyl (2S,4E)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.036

Methyl (2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.012

Methyl (2S,4EZ)-4-(methoxyimino)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.10

[0229] b) Functional Assay No. 1: Inhibition of oxytocin mediatedCa²⁺-mobilization by FLIPR® (Fluorimetric Imaging Plate Reader)

[0230] The action of OT on the OT-receptor triggers a complex cascade ofevents in the cell which leads to an increase in the intra-cytoplasmicCa²⁺ concentration. This increase in Ca²⁺ concentration results fromboth calcium release from the sarcoplasmic reticulum (calcium stores)into the cytoplasm and from calcium influx from the extracellular spacethrough Ca²⁺ channels. This Ca²⁺ mobilization into the cytoplasmtriggers the contractile machinery of the myometrial cells which leadsto uterine contractions (see Gimpl G. and Fahrenholz, F. PhysiologicalReviews 2001, 81, 629-683 as well as Mitchell, B. F. and Schmid, B. JSoc. Gynecol. Invest. 2001, 8,122-33).

[0231] This assay allows the measurement of the inhibition of OT/OT-Rmediated calcium mobilization by test compounds of formula (I).

[0232] FLIPR® is a fluorimetric imaging device using a laser (Argon-ionlaser) for simultaneous illumination and reading (cooled CCD camera) ofeach well of a 96-well-plate, thus enabling rapid measurements on alarge number of samples.

[0233] Preparing the plates: FLIPR-plates were pre-coated with PLL(Poly-L-Lysine) 10 μg/ml+0.1% gelatine to attach HEK293EBNA cells (HumanEmbryonic Kidney cells expressing the hOT receptor) and incubated for 30min up to 2 days at 37° C. The cells were plated out into 96-well-plates(60000 cells/well).

[0234] Labelling with fluo-4: 50 μg of fluo-4 (Ca2+ sensitivefluorescent dye) were dissolved in 20 μl pluronic acid (20% in DMSO).The dissolved fluo-4 was then diluted in 10 ml DMEM (Dubecco's MinimalEssential Medium)-F12 culture medium. The plates were washed one timewith DMEM-F12 medium. 100 μl of the fluo-4 containing-DMEM-F12 mediumwere added to the HEK-cells which were incubated for 1.5-2 h in thisfluorescent medium. Fluo-4 is taken up by the cytoplasm of the cells.

[0235] Buffer: 145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM Hepes, 10 mMGlucose, EGTA (Ethylene-bis oxyethylene nitrilo tetraacetic acid). ThepH was adjusted to 7.4.

[0236] Performance of the assay: A minimum of 80 μl/well of compounds offormula (I) (5×) in the above buffer (1×) were prepared(96-well-plates). The compounds of formula (I) were added to the96-well-plates at different concentrations (30 μM, 10 μM, 1 μM, 300 nM,100 nM, 10 nM, 1 nM, 100 pM, 10 pM). OT was added at a concentration of40 nM.

[0237] The relative fluorescence of Fluo-4 (λ_(ex)=488 nm, λ_(em)=590nm) is then measured by the FLIPR in presence or absence of compounds offormula (1). The fluorescence of the marker being sensitive to theamount of Ca²⁺, the Ca²⁺ movements can be detected. Then, the ability ofcompounds of formula (I) to antagonize the oxytocin-inducedintracellular Ca²⁺-mobilization mediated by the oxytocin receptor may bedetermined.

[0238] The activities of the pyrrolidine derivatives according toformula (1) were assessed using the above described in vitro biologicalassay. Representative values for some example compounds are given inTable 2. The values refer to the concentration of the test compoundsaccording to formula (I) necessary to antagonize by 50% the OT/OTRintracellular Ca²⁺-mobilization. From the values, it can be derived thatsaid example compounds according to formula (I) do exhibit a significantactivity as oxytocin receptor antagonists. TABLE 2 Inhibition of Ca²⁺mobiliation, hOT-R Structure IUPAC-Name IC₅₀ (μM)

Methyl (2S,4E)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.015

Methyl (2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.015

[0239] c) Functional Assay No. 2: Inhibition of IP3 (InositolTri-Phosphate)-Synthesis in HEK/EBNA-OTR cells

[0240] The interaction of OT on the OT-receptor leads to the IP3synthesis, IP3 being a second messenger for the Ca²⁺ release fromsarcoplasmic reticulum, involved in the uterine contraction triggeringprocess (see Mitchell, B. F. and Schmid, B. J. Soc. Gynecol. Invest.2001, 8,122-33).

[0241] This assay can be used to show the inhibition of the OT/OT-Rmediated IP3 synthesis by using test compounds of formula (I).

[0242] Stimulation of the cells: HEK/EBNA OTR (rat or human) cells areplated out-into costar 12-well plates, and equilibrated for 15-24 h with4 μCi/ml radiolabelled [³H]-Inositol with 1% FCS (0.5 ml/well) andwithout inositol supplement. The medium containing the label isaspirated. DMEM medium (without FCS, inositol), 20 mM Hepes(4-(2-hydroxyethyl)-1-piperazine-ethane-sulphonic acid), 1 mg/ml BSAcontaining 10 mM LiCl (freshly prepared), are added and incubated for10-15 min at 37° C. The agonist (i.e. oxytocin used at a concentrationof 10 nM) and the antagonists (i.e. the tests compounds of formula (I)can be used in a concentration of 10 μM, 1 μM, 300 nM, 100 nM, 10 nM, 1nM, 100 pM, 10 pM, 3 pM) can be added at the required time (15-45 min),followed by aspiration of the medium. In the presence of OT, theradiolabelled inositol is converted to radiolabelled IP3. AntagonizingOT at the OT-receptor inhibits the IP3 formation.

[0243] The amount of the radiolabelled IP3 may be determined through theensuing work-up. The reaction is stopped with 1 ml STOP-solution (i.e.0.4 M perchloric acid), and let sit for 5-10 min at Room Temperature.Then, 0.8 ml are transferred into tubes containing 0.4 ml ofneutralizing solution (0.72 M KOH/0.6M KHCO₃), and the tubes vortexedand kept in the cold at least for 2 h.

[0244] Separation of IP's: The samples are spun in a table topcentrifuge at 3000-4000 rpm for 15 min. 1 ml of the supernatant istransferred to new tubes containing 2.5 ml H₂O. Packed resin (DowexAG1×8) is equilibrated with 20 ml H₂O, and the whole samples are pouredonto the chromatography columns, thus separating the mixture. To removefree inositol, two washes with 10 ml H₂O are carried out.

[0245] Elution of total IP's: Elution is achieved using 3 ml 1M ammoniumformate/0.1M formic acid. The eluant is collected in scintillationcounting tubes, after the addition of 7 ml of scintillation liquid. Theamount of [³H]-IP3 is determined by a scintillating counter.

[0246] The ability of compounds of formula(I) to effectively antagonizeoxytocin-induced IP3synthesis mediated by the oxytocin receptor, can beassessed using the above described in vitro biological assay. TABLE 3Inhibition of IP3- synthesis, ratOT-R Structure IUPAC-Name IC₅₀ (μM)

Methyl (2S,4E)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.077

Methyl (2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate 0.023

[0247] d) In Vivo Model for Inhibtion of Uterine Contractions

[0248] The assay evaluates the biological effect of tested compounds inan in vivo model of preterm labor, premature birth.

[0249] Non-pregnant Charles River CD (SD) BR female rats (9-10 weeksold, 200-250 g) were treated at 18 and 24 hours before the experimentwith 250 μg/kg, i.p. diethylstilbestrol (DES). For the assay, the animalwas anaesthetised with urethane (1.75 g/kg, i.p.) and placed on ahomeothermic operating table. The trachea was isolated and cannulatedwith a suitable polyethylene (PE) tubing. A midline incision at thehypogastrium level was made and one uterine horn exposed, its cephalicend cannulated with a PE240 tubing and, after filling the internalcavity with 0.2 ml of sterile physiological saline, connected to a“Gemini” amplifying/recording system via a P23ID Gould Statham pressuretransducer.

[0250] One jugular vein was isolated, cannulated with a PE60 tubing andconnected to a butterfly needle to provide an i.v. route ofadministration of the test compounds via a dispensing syringe.

[0251] In the case of intraduodenal administration of the testcompounds, the duodenum can be isolated and similarly cannulated througha small incision in its wall.

[0252] One carotid artery was also isolated and cannulated with PE60catheter and connected to a suitable syringe for blood samplecollection.

[0253] After a stabilization period and throughout the experiment, thesame dose of oxytocin was repeatedly injected intravenously at 30-minintervals. When reproducible contractile responses of the uterus to thesame OT stimulus (selected dose of oxytocin) were obtained, the dose ofthe test compound or of the reference (vehicle) was administered.Further injection cycles of the same dose of oxytocin, were continued(OT injections at 30-min intervals) for a suitable time after treatmentto assess the inhibitory effects and the reversibility of these effects.

[0254] The contractile response of the uterus to oxytocin was quantifiedby measuring the intrauterine pressure and the number of contractions.The effect of the reference and test compounds was evaluated bycomparing pre- and post-treatment pressure values. In addition, at 2,30, 90 and 210 minutes after test compound administration, a 0.5-mlblood sample was withdrawn from the cannulated carotid artery of eachexperimental animal. Plasma was obtained by standard laboratoryprocedure and the resulting samples were stored at −20° C.

[0255] The activities of the pyrrolidine derivatives of formula (I) maybe assessed using the above described in vivo biological assay.Representative values for one example compound are given in Table 4. Thevalues refer to the capacity of the example compound according toformula (I) to effectively antagonize oxytocin-induced uterinecontractions in the rat. From the values shown in Table 4 it may bederived that said example test compound according to formula (I) doesexhibit a significant activity as tocolytic, i.e. uterine-relaxing,agent. TABLE 4 Route of % Reduction of administration/ Uterine DoseStructure IUPAC-Name Vehicle Contraction (mg/kg)

Methyl (2S,4Z)-4- (methoxyimino)-1-[(2′- methyl[1,1′-biphenyl]-4-yl)carbonyl]-2- pyrrolidinecarboxylate intravenous; PEG400/saline 50:50;5 ml/kg infusion −35.4 ± 7.0 −49.0 ± 6.5 −51.8 ± 9.2 1 3 10 

1. Use of a pyrrolidine ester according to formula (I)

as well as its geometrical isomers, its optically active forms asenantiomers, diastereomers and its racemate forms, as well aspharmaceutically acceptable salts thereof, wherein X is selected fromthe group consisting of CR⁶R⁷, NOR⁶, NNR⁶R⁷; R is selected from thegroup comprising or consisting of C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, saturated or unsaturated 3-8-membered cycloalkyl which maycontain 1 to 3 heteroatoms selected of N, O, S, aryl, heteroaryl,C₁-C₆-alkyl aryl, C₁-C₆-alkyl heteroaryl, C₁-C₆-alkyl-saturated orunsaturated 3-8-membered cycloalkyl. R¹ is selected from the groupcomprising or consisting of C₁-C₆-alkyl, aryl, heteroaryl, saturated orunsaturated 3-8-membered cycloalkyl, acyl, C₁-C₆-alkyl aryl, C₁-C₆-alkylheteroaryl, said cycloalkyl or aryl or heteroaryl groups may be fusedwith 1-2 further cycloalkyl or aryl or heteroaryl group; R², R³, R⁴ andR⁵ are independently selected from each other from the group consistingof hydrogen, halogen, C₁-C₆-alkyl; R⁶ and R⁷ are independently selectedfrom the group comprising or consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, C₁-C₆ thioalkoxy, halogen, cyano,nitro, acyl, alkoxycarbonyl, aminocarbonyl, saturated or unsaturated3-8-membered cycloalkyl which may contain 1 to 3 heteroatoms selected ofN, O, S, aryl, heteroaryl, C₁-C₆-alkyl aryl, C₁-C₆-alkyl heteroaryl,C₁-C₆-alkyl-saturated or unsaturated 3-8-membered cycloalkyl; or R⁶, R⁷could form together with the N atom to which they are attached a 3-8membered saturated or unsaturated heterocyclic ring which may contain1-2 further heteroatoms selected from N, S and O and which is optionallyfused with an aryl, heteroaryl or 3-8 membered saturated or unsaturatedcycloalkyl ring; for the preparation of a pharmaceutical composition forthe treatment and/or prevention of premature labor, premature birth anddysmenorrhea.
 2. Use according to claim 1, wherein X is NOR⁶ and R⁶ isselected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, acyl, aryl, heteroaryl, saturated or unsaturated3-8-membered cycloalkyl, C₁-C₆-alkyl aryl, C₁-C₆-alkyl heteroaryl, saidcycloalkyl or aryl or heteroaryl groups may be fused with 1-2 furthercycloalkyl or aryl or heteroaryl groups.
 3. Use according to claim 2,wherein R⁶ is H or CH₃.
 4. Use according to any of preceding claims,wherein R¹ is a C₁-C₆-alkyl, C₂-C₆-alkenyl, C₂-C₆-alkynyl, aryl,heteroaryl, saturated or unsaturated 3-8-membered cycloalkyl.
 5. Useaccording to claim 4, wherein R¹ is a biphenyl
 6. Use according to anyof preceding claims, wherein X is NOR⁶, R¹ is H, a C₁-C₆-alkyl, or arylor C₁-C₆-alkyl aryl group and R¹ is selected from C₁-C₆-alkyl; aryl orC₁-C₆alkyl aryl.
 7. Use according to claim 6, wherein R⁶ is methyl, R isa C₁-C₆-alkyl group, in particular a methyl group, and R¹ is a biphenyl.8. Use of a pyrrolidine derivative according to any of claims 1 to 7 forthe preparation of a pharmaceutical composition for the modulation ofthe oxytocin receptor.
 9. Use according to claim 8, wherein saidmodulation consists in the blocking of the oxytocin receptor or inantagonising the binding of oxytocin to its receptor.
 10. Use of apyrrolidine derivative according to any of claims 1 to 7 for thepreparation of a pharmaceutical composition for the treatment orprevention of disorders mediated by the oxytocin receptor.
 11. Apyrrolidine ester compound of formula (I′),

wherein R is selected from C₁-C₆ alkyl, C₁-C₆ alkyl aryl, C₁-C₆ alkylheteroaryl, 3-8-membered cycloalkyl and R¹ is selected from a1,1′-biphenyl, a pyridinyl-phenyl or a pyrimidinyl-phenyl group.
 12. Apyrrolidine ester compound according to claim 11, wherein R is a methylgroup.
 13. A pyrrolidine ester compound according to any of claims 11 or12, wherein R¹ is a 1,1′-biphenyl group.
 14. A pyrrolidine estercompound according to any of claims 11 to 13, wherein R¹ is a1,1′-biphenyl group which is substituted by 1 or 2 moieties selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, halogen, CN. 15.A pyrrolidine derivative according to any of claims 11 to 14, selectedfrom the group consisting of: Methyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-([1,1′-biphenyl]-4-ylcarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4E)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylateMethyl(2S,4Z)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4Z)-1-[(2′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(2′-chloro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(2′-cyano[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl (2S,4EZ)-4-(methoxyimino)-1{[2′-(trifluoromethyl)[1,1′-biphenyl]-4-yl]carbonyl}-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(2′-methoxy[1,1′-biphenyl]4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(2′,6′-dimethyl[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(2′,3-dimethyl[1,1′-biphenyl]4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(3-methyl[1,1′-biphenyl]-4-yl)carbonyl]4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(3′,4′-dichloro[1,1′-biphenyl]-4-yl)carbonyl]-4-(methoxyimino)-2-pyrrolidinecarboxylateEthyl(2S,4EZ)-1-([1,1′-biphenyl]-4-ylcarbonyl)-4-(methoxyimino)-2-pyrrolidinecarboxylatesec-butyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]2-pyrrolidinecarboxylateCyclopentyl(2S,4EZ)-4-(methoxyimino)-1-[(2′-methyl[1,1′-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-1-[(4′-fluoro[1,1′-biphenyl]-4-yl)carbonyl]4-(methoxyimino)-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-4-(methoxyimino)-1-[4-(5-pyrimidinyl)benzoyl]-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-4-(methoxyimino)-1-[4-(2-pyridinyl)benzoyl]-2-pyrrolidinecarboxylateMethyl(2S,4EZ)-4-(methoxyimino)-1-[4-(3-methyl-2-pyridinyl)benzoyl]-2-pyrrolidinecarboxylate16. A pyrrolidine derivative according to any of claims 11 to 15 for useas a medicament.
 17. A pharmaceutical composition containing at leastone pyrrolidine derivative according to any of claims 11 to 14 and apharmaceutically acceptable carrier, diluent or excipient thereof.
 18. Aprocess of preparing a compound according to any of claims 11 to 15,comprising the step of reacting a compound of formula (III)

wherein X, R²-R⁵ are H and R are as above defined, with a carboxylicacid or acyl chloride of formulae (IVa) or (IVb)

wherein R¹ is as above defined.